Nicolle Packer

Configurations of glycosidic phosphates of lipopolysaccharide from Salmonella minnesota R595

Publisher: American Chemical Society (ACS)

Date: 07-12-1982

DOI: 10.1021/BI00268A040

Abstract: The anomeric configurations of the reducing terminal glucosamine and 4-amino-4-deoxy-L-arabinose phosphates in lipopolysaccharide from Salmonella minnesota R595 have been determined by nuclear magnetic resonance. Chemical shifts for the anomeric protons were obtained by selective decoupling of the phosphorus spectrum and proton-proton coupling constants by polarization transfer from protons to phosphorus. In both cases, the phosphate is attached to the sugar in an axial orientation.

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis

Publisher: Springer Science and Business Media LLC

Date: 11-2021

DOI: 10.1038/S41592-021-01309-X

Abstract: Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometry based glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O -glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved ‘high-coverage’ and ‘high-accuracy’ glycoproteomics search solutions. This study concludes that erse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

Introducing Glycoproteomics, a new section of PROTEOMICS

Publisher: Wiley

Date: 12-2006

DOI: 10.1002/PMIC.200690140

Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1074/JBC.M116.735258

Light-Emitting Diode Excitation for Upconversion Microscopy: A Quantitative Assessment

Publisher: American Chemical Society (ACS)

Date: 16-09-2020

DOI: 10.1021/ACS.NANOLETT.0C02697

Mucin-type O-glycosylation - Putting the pieces together

Publisher: Wiley

Date: 15-12-2010

DOI: 10.1111/J.1742-4658.2009.07429.X

Abstract: The O-glycosylation of Ser and Thr by N-acetylgalactosamine-linked (mucin-type) oligosaccharides is often overlooked in protein analysis. Three characteristics make O-linked glycosylation more difficult to analyse than N-linked glycosylation, namely: (a) no amino acid consensus sequence is known (b) there is no universal enzyme for the release of O-glycans from the protein backbone and (c) the density and number of occupied sites may be very high. For significant biological conclusions to be drawn, the complete picture of O-linked glycosylation on a protein needs to be determined. This review specifically addresses the analytical approaches that have been used, and the challenges remaining, in the characterization of both the composition and structure of mucin-type O-glycans, and the determination of the occupancy and heterogeneity at each amino acid attachment site.

MUC5B glycosylation in human saliva reflects blood group and secretor status

Publisher: Oxford University Press (OUP)

Date: 06-04-2005

DOI: 10.1093/GLYCOB/CWI059

Abstract: This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from in iduals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose olyacrylamide composite gel electrophoresis (SDS-AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive beta-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O in iduals into larger population (29 in iduals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 in iduals. The remaining six in iduals were characterized by a high degree of sialylation. These in iduals were assigned as nonsecretors, whereas blood group-expressing in iduals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Le(a)) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between in iduals and that some of this variation is owing to blood group and secretor status.

Periconception onset diabetes is associated with embryopathy and fetal growth retardation, reproductive tract hyperglycosylation and impaired immune adaptation to pregnancy

Publisher: Springer Science and Business Media LLC

Date: 02-2018

DOI: 10.1038/S41598-018-19263-8

Abstract: Diabetes has been linked with impaired fertility but the underlying mechanisms are not well defined. Here we use a streptozotocin-induced diabetes mouse model to investigate the cellular and biochemical changes in conceptus and maternal tissues that accompany hyperglycaemia. We report that streptozotocin treatment before conception induces profound intra-cellular protein β- O -glycosylation ( O -GlcNAc) in the oviduct and uterine epithelium, prominent in early pregnancy. Diabetic mice have impaired blastocyst development and reduced embryo implantation rates, and delayed mid-gestation growth and development. Peri-conception changes are accompanied by increased expression of pro-inflammatory cytokine Trail , and a trend towards increased Il1a , Tnf and Ifng in the uterus, and changes in local T-cell dynamics that skew the adaptive immune response to pregnancy, resulting in 60% fewer anti-inflammatory regulatory T-cells within the uterus-draining lymph nodes. Activation of the heat shock chaperones, a mechanism for stress deflection, was evident in the reproductive tract. Additionally, we show that the embryo exhibits elevated hyper- O -GlcNAcylation of both cytoplasmic and nuclear proteins, associated with activation of DNA damage (ɣH2AX) pathways. These results advance understanding of the impact of peri-conception diabetes, and provide a foundation for designing interventions to support healthy conception without propagation of disease legacy to offspring.

Understanding cellular glycan surfaces in the central nervous system

Publisher: Portland Press Ltd.

Date: 17-12-2019

DOI: 10.1042/BST20180330

Abstract: Glycosylation, the enzymatic process by which glycans are attached to proteins and lipids, is the most abundant and functionally important type of post-translational modification associated with brain development, neurodegenerative disorders, psychopathologies and brain cancers. Glycan structures are erse and complex however, they have been detected and targeted in the central nervous system (CNS) by various immunohistochemical detection methods using glycan-binding proteins such as anti-glycan antibodies or lectins and/or characterized with analytical techniques such as chromatography and mass spectrometry. The glycan structures on glycoproteins and glycolipids expressed in neural stem cells play key roles in neural development, biological processes and CNS maintenance, such as cell adhesion, signal transduction, molecular trafficking and differentiation. This brief review will highlight some of the important findings on differential glycan expression across stages of CNS cell differentiation and in pathological disorders and diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia and brain cancer.

Multidimensional fractionation is a requirement for quantitation of golgi-resident glycosylation enzymes from cultured human cells

Publisher: American Chemical Society (ACS)

Date: 24-12-2015

DOI: 10.1021/PR500785F

Abstract: Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation

Publisher: Springer Science and Business Media LLC

Date: 12-11-2015

DOI: 10.1007/S10719-015-9627-1

Changes in dietary fiber intake in mice reveal associations between colonic mucin O-glycosylation and specific gut bacteria

Publisher: Informa UK Limited

Date: 29-09-2020

DOI: 10.1080/19490976.2020.1802209

Characterisation of individual N-and O-linked glycosylation sites using Edman degradation

Publisher: Elsevier

Date: 1995

DOI: 10.1016/S1080-8914(06)80013-X

Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G

Publisher: MDPI AG

Date: 04-03-2015

DOI: 10.3390/BIOM5031832

GlycoStore: a database of retention properties for glycan analysis

Publisher: Oxford University Press (OUP)

Date: 20-04-2018

DOI: 10.1093/BIOINFORMATICS/BTY319

Abstract: GlycoStore is a curated chromatographic, electrophoretic and mass-spectrometry composition database of N-, O-, glycosphingolipid (GSL) glycans and free oligosaccharides associated with a range of glycoproteins, glycolipids and biotherapeutics. The database is built on publicly available experimental datasets from GlycoBase developed in the Oxford Glycobiology Institute and then the National Institute for Bioprocessing Research and Training (NIBRT). It has now been extended to include recently published and in-house data collections from the Bioprocessing Technology Institute (BTI) A*STAR, Macquarie University and Ludger Ltd. GlycoStore provides access to approximately 850 unique glycan structure entries supported by over 8500 retention positions determined by: (i) hydrophilic interaction chromatography (HILIC) ultra-high performance liquid chromatography (U/HPLC) and reversed phase (RP)-U/HPLC with fluorescent detection (ii) porous graphitized carbon (PGC) chromatography in combination with ESI-MS/MS detection and (iii) capillary electrophoresis with laser induced fluorescence detection (CE-LIF). GlycoStore enhances many features previously available in GlycoBase while addressing the limitations of the data collections and model of this popular resource. GlycoStore aims to support detailed glycan analysis by providing a resource that underpins current workflows. It will be regularly updated by expert annotation of published data and data obtained from the project partners. www.glycostore.org Supplementary data are available at Bioinformatics online.

Sequential Analysis of N- and O-Linked Glycosylation of 2D-PAGE Separated Glycoproteins

Publisher: American Chemical Society (ACS)

Date: 07-09-2002

DOI: 10.1021/PR025538D

Abstract: A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha2-HS-glycoprotein, serotransferrin, alpha1-antitrypsin, and alpha1-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.

Analyzing glycoproteins separated by two-dimensional gel electrophoresis

Publisher: Wiley

Date: 05-1998

DOI: 10.1002/ELPS.1150190613

Abstract: Two-dimensional (2-D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as 'trains' of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the 'rules' which govern the migration of glycoproteins in 2-D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N-linked oligosaccharides) or chemically (beta-elimination of O-linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic removal of the N-linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O-linked oligosaccharides. These approaches have been proved on 1-D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2-D PAGE preparative narrow range (pH 4.5-5.5) blots of human plasma: alpha2-HS glycoprotein (human fetuin) and alpha1-antitrypsin (alpha1-protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity ( ersity of structures) of the glycosylation contribute to the separation of protein isoforms in 2-D PAGE.

Validation of the curation pipeline of UniCarb-DB: Building a global glycan reference MS/MS repository

Publisher: Elsevier BV

Date: 2014

DOI: 10.1016/J.BBAPAP.2013.04.018

Abstract: The UniCarb-DB database is an emerging public glycomics data repository, containing over 500 tandem mass spectra (as of March 2013) of glycans released from glycoproteins. A major challenge in glycomics research is to provide and maintain high-quality datasets that will offer the necessary ersity to support the development of accurate bioinformatics tools for data deposition and analysis. The role of UniCarb-DB, as an archival database, is to provide the glycomics community with open-access to a comprehensive LC MS/MS library of N- and O- linked glycans released from glycoproteins that have been annotated with glycosidic and cross-ring fragmentation ions, retention times, and associated experimental metadata descriptions. Here, we introduce the UniCarb-DB data submission pipeline and its practical application to construct a library of LC-MS/MS glycan standards that forms part of this database. In this context, an independent consortium of three laboratories was established to analyze the same 23 commercially available oligosaccharide standards, all by using graphitized carbon-liquid chromatography (LC) electrospray ionization (ESI) ion trap mass spectrometry in the negative ion mode. A dot product score was calculated for each spectrum in the three sets of data as a measure of the comparability that is necessary for use of such a collection in library-based spectral matching and glycan structural identification. The effects of charge state, de-isotoping and threshold levels on the quality of the input data are shown. The provision of well-characterized oligosaccharide fragmentation data provides the opportunity to identify determinants of specific glycan structures, and will contribute to the confidence level of algorithms that assign glycan structures to experimental MS/MS spectra. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Relative versus absolute quantitation in disease glycomics

Publisher: Wiley

Date: 24-03-2015

DOI: 10.1002/PRCA.201400184

Abstract: The glycome of a diagnostic biological material such as blood, urine, saliva, tissue, or cell cultures comprises of a vast array of structurally distinct glycans attached to the protein complement. Aberrant glycan structures and distributions result from changes in specific glycosyltransferase activities and have different biological significance, making proper quantitation of glycans highly important. In this review, common HPLC/CE and LC-MS/MS-based methods for glycomics, their advantages and disadvantages, will be discussed with respect to the main quantitative strategies. With the increasing interest in absolute quantitation for glycomics, we discuss absolute and relative glycome quantitation and how it affects the resulting conclusions drawn from glycomics studies. We argue that while absolute quantitation of glycomes may be attractive for some areas of clinical glycomics, relative quantitation of glycans remains the most informative and time/cost-effective method to obtain biological insight into the regulation of the cellular glycosylation machinery and the synthesis of the resultant glycan structures in most research questions due to the enzymatic relatedness of the biosynthesized glycans. Recent developments in multiplexing of glycomes by the introduction of stable isotopic labeling of glycans show promise for providing another level of information to the existing benefits of relative quantitation.

Sweating the small stuff

Publisher: Oxford University Press (OUP)

Date: 17-11-2016

DOI: 10.1093/GLYCOB/CWV102

Abstract: There is increasing evidence that secretory fluids such as tears, saliva and milk play an important role in protecting the human body from infection via a washing mechanism involving glycan-mediated adhesion of potential pathogens to secretory glycoproteins. Interaction of sweat with bacteria is well established as the cause of sweat-associated malodor. However, the role of sweat glycoproteins in microbial attachment has received little, if any, research interest in the past. In this review, we demonstrate how recent published studies involving high-throughput proteomic analysis have inadvertently, and fortuitously, exposed an abundance of glycoproteins in sweat, many of which have also been identified in other secretory fluids. We bring together research demonstrating microbial adhesion to these secretory glycoproteins in tears, saliva and milk and suggest a similar role of the sweat glycoproteins in mediating microbial attachment to sweat and/or skin. The contribution of glycan-mediated microbial adhesion to sweat glycoproteins, and the associated impact on sweat derived malodor and pathogenic skin infections are unchartered new research areas that we are beginning to explore.

MALDI mass spectrometry imaging of N-glycans on tibial cartilage and subchondral bone proteins in knee osteoarthritis

Publisher: Wiley

Date: 06-2016

DOI: 10.1002/PMIC.201500461

Abstract: Magnetic resonance imaging (MRI) is a non-invasive technique routinely used to investigate pathological changes in knee osteoarthritis (OA) patients. MRI uniquely reveals zones of the most severe change in the subchondral bone (SCB) in OA, called bone marrow lesions (BMLs). BMLs have diagnostic and prognostic significance in OA, but MRI does not provide a molecular understanding of BMLs. Multiple N-glycan structures have been observed to play a pivotal role in the OA disease process. We applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of N-glycans to formalin-fixed paraffin-embedded (FFPE) SCB tissue sections from patients with knee OA, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was conducted on consecutive sections to structurally characterize and correlate with the N-glycans seen by MALDI-MSI. The application of this novel MALDI-MSI protocol has enabled the first steps to spatially investigate the N-glycome in the SCB of knee OA patients.

Alcoholic liver disease alters glycosylation-associated gene expression

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1016/S0168-8278(14)60484-0

MIRAGE: The minimum information required for a glycomics experiment

Publisher: Oxford University Press (OUP)

Date: 20-03-2014

DOI: 10.1093/GLYCOB/CWU018

Multiple O-glycoforms on the spore coat protein sp96 in Dictyostelium discoideum. Fuc(α1-3)GlcNAc-α-1-P-Ser is the major modification

Publisher: Elsevier BV

Date: 04-2000

DOI: 10.1074/JBC.275.16.12164

FKRP-dependent glycosylation of fibronectin regulates muscle pathology in muscular dystrophy

Publisher: Springer Science and Business Media LLC

Date: 19-05-2021

DOI: 10.1038/S41467-021-23217-6

Abstract: The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these erse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG’s glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.

Moditied glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei

Publisher: Wiley

Date: 15-08-1998

DOI: 10.1046/J.1432-1327.1998.2560119.X

Abstract: Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.

Toolboxes for a standardised and systematic study of glycans

Publisher: Springer Science and Business Media LLC

Date: 2014

DOI: 10.1186/1471-2105-15-S1-S9

19th International Meeting of the European Society of Gynecological Oncology (ESGO 2015)

Publisher: BMJ

Date: 10-2015

DOI: 10.1097/01.IGC.0000473498.85773.6E

Graphitized Carbon LC−MS Characterization of the Chondroitin Sulfate Oligosaccharides of Aggrecan

Publisher: American Chemical Society (ACS)

Date: 06-04-2007

DOI: 10.1021/AC0622227

Abstract: A novel in-gel endoglycosidase technique to study oligosaccharides with graphitized carbon LC-MS has revealed differences in the sulfation profile between the linkage and repeat regions of chondroitin sulfate on aggrecan. Bovine articular cartilage aggrecan was isolated in a composite agarose PAGE gel or diluted in ammonium acetate buffer and was digested overnight with chondroitinase ABC. Including a chemical release/reduction protocol after digestion, we could separate and detect three differentially sulfated chondroitin sulfate disaccharides of the repeat region (DeltaUA1-3GalNAc0/4/6S-ol) from the three differentially sulfated linkage region hexasaccharides (DeltaUA1-3GalNAc0/4/6Sbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylitol). Graphitized carbon LC-MS in the negative ion mode was able to resolve isomeric disaccharides and linkage region hexasaccharides. Specific MS2 and MS3 enabled us to confirm the sulfate location on all oligosaccharides by comparing their fragmentation with sulfated disaccharide standards. The presence of unsulfated, 6-sulfated, and 4-sulfated linkage regions was correlated with positive Western blot staining with the respective CS linkage region neoepitope antibodies (1B5, 3B3, 2B6) on digested aggrecan. Our strategy of examining linkage region and repeat region profiles is applicable to screening GAGs from various biological s les in order to detect differences between normal and disease states.

Characterization of human plasma glycoproteins separated by two-dimensional gel electrophoresis

Publisher: Springer Science and Business Media LLC

Date: 1996

DOI: 10.1038/NBT0196-66

Abstract: Purification of protein isoforms for the characterization of post-translational modifications, such as glycosylation, can be laborious and demanding. We report a means of determining monosaccharide composition and the identity of glycoproteins from a single spot on a two-dimensional (2-D) gel. The sensitivity of the method depends on the degree of glycosylation of the protein. We show that bovine fetuin can be analyzed and identified at the level of 100 pmol. 2-D reference maps enable quick identification of glycoprotein isoforms, and the nature of glycosylation differences. Human sera glycoforms were isolated by micropreparative 2-D PAGE using a narrow-range immobilized pH gradient. Single spots excised from one polyvinylidene difluoride blot of a 2-D gel were used sequentially for sialic acid analysis, neutral and amino sugar analysis, and finally amino acid analysis. The glycosylation variations in isoforms of human fetuin and alpha-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.

UniCarbKB: Putting the pieces together for glycomics research

Publisher: Wiley

Date: 19-09-2011

DOI: 10.1002/PMIC.201100302

Abstract: Despite the success of several international initiatives the glycosciences still lack a managed infrastructure that contributes to the advancement of research through the provision of comprehensive structural and experimental glycan data collections. UniCarbKB is an initiative that aims to promote the creation of an online information storage and search platform for glycomics and glycobiology research. The knowledgebase will offer a freely accessible and information-rich resource supported by querying interfaces, annotation technologies and the adoption of common standards to integrate structural, experimental and functional data. The UniCarbKB framework endeavors to support the growth of glycobioinformatics and the dissemination of knowledge through the provision of an open and unified portal to encourage the sharing of data. In order to achieve this, the framework is committed to the development of tools and procedures that support data annotation, and expanding interoperability through cross-referencing of existing databases. Database URL: www.unicarbkb.org.

The use of solid-phase extraction with graphitised carbon for the fractionation and purification of sugars

Publisher: Elsevier BV

Date: 06-1999

DOI: 10.1016/S0008-6215(99)00130-5

Host mucin glycosylation plays a role in bacterial adhesion in lungs of individuals with cystic fibrosis

Publisher: Informa UK Limited

Date: 10-2013

DOI: 10.1586/17476348.2013.837752

Abstract: Malfunction of the cell surface glycoprotein, cystic fibrosis transmembrane conductance regulator, is the molecular hallmark of cystic fibrosis (CF), causing salt imbalance across the lung epithelium and biochemical and biophysical alterations of the mucous secretion and airway surfaces. Abnormal glycosylation of both secreted and membrane-tethered airway mucins in CF hosts are reported by a substantial body of literature and correlates with bacterial infection and inflammation in CF airways, features that are linked to the CF pathology. It is established that Pseudomonas aeruginosa and other CF-typic bacteria use the altered host mucin glycosylation as receptors for adhesion by dedicated lectins and adhesins recognizing an array of the aberrantly expressed glycan determinants. This review aims to describe the aberrant mucin glycosylation phenotype observed in CF airways relative to the non-CF equivalent by summarizing the wealth of literature on this topic. The possible causes and effects of altered glycosylation in the respiratory system are discussed. Specific attention is given to the adhesion mechanisms of the opportunistic P. aeruginosa, which utilizes the molecular alterations of the lung to gain access to the normally sterile airways. Finally, the emerging glycosylation-based therapeutics that show promising potential for reducing bacterial infection in in iduals with CF by molecular mimicry mechanisms are discussed.

Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1074/JBC.M114.631622

Comparing the chemical composition of dietary fibres prepared from sugarcane, psyllium husk and wheat dextrin

Publisher: Elsevier BV

Date: 11-2019

DOI: 10.1016/J.FOODCHEM.2019.125032

Abstract: A dietary fibre prepared from sugarcane stalk was compared with psyllium husk and wheat dextrin. In contrast to the other dietary fibres, sugarcane fibre was found to contain significant amounts of insoluble dietary fibre (73-86%), lignin (18.66-20.23%), and rare minerals such as chromium (0.67-2.54 mg/100 g) and manganese (1.07-2.34 mg/100 g). Analysis of the ethanol extract also detected compounds with antioxidant activity. Characterisation of five sugarcane fibres prepared from selected strains, harvest periods (growth or storage phase), and processing conditions showed these factors influenced the final composition. Furthermore, using in vitro digestion, we found that potassium, magnesium, chromium, and zinc in were bioaccessible in sugarcane s les. Also, sodium was shown to bind to the sugarcane fibre potentially indicating bile salt binding activity. Results from this study support the use of sugarcane as a source of dietary fibre in functional foods.

Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand

Publisher: American Chemical Society (ACS)

Date: 15-09-2016

DOI: 10.1021/ACS.ANALCHEM.6B02191

Abstract: We describe the application of a synthetically developed tetradentate β-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.

Integrating Quickbird Multi-Spectral Satellite and Field Data: Mapping Bathymetry, Seagrass Cover, Seagrass Species and Change in Moreton Bay, Australia in 2004 and 2007

Publisher: MDPI AG

Date: 06-01-2011

DOI: 10.3390/RS3010042

Better together – relative retention time plus spectral matching improves automated glycan characterization using PGC-nLC-IT-ESI-MS/MS

Publisher: Cold Spring Harbor Laboratory

Date: 04-08-2021

DOI: 10.1101/2021.08.04.451140

Abstract: Porous Graphitized Carbon nano-liquid chromatography tandem mass spectrometry (PGC-nLC-MS/MS) is a glycomics technique with the unique capacity to differentiate isobaric glycans. The lack of suitable software tools integrating chromatography and MS-information delivered by PGC-nLC-MS/MS has been limiting fast and robust glycan identification and quantitation. We report a LC-system-independent strategy called GlycoRRT that combines relative retention time (RRT) and negative ion fragment spectra analyses for isobaric structure-specific glycomics of PGC-nLC-MS/MS data. The GlycoRRT toolset is fully customizable and easily adaptable enabling semi-automated high-throughput structural assignments. The current library contains over 200 entries and their in idual meta-data (MS instrumentation, experimental conditions, retention times, fragmentation profiles and glycan structural diagnostic ion features) relevant for reliable data analyses. The GlycoRRT workflow was employed to map the N - and O - glycome in blood group matched human plasma and urine as well as decipher Immunoglobulin (IgG) glycosylation features from 13 different animal species. We have also developed visualization tools to enable a consistent, reliable, and reproducible analysis of large sets of multidimensional PGC-nLC-MS/MS glycomics data. This comprehensive glycan resource provides the glycan map of human and animal species, will serve as a reference in dissecting the role of glycans in host pathogen interaction and zoonotic disease transmission.

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of SerumN- andO-Glycopeptide Data

Publisher: Cold Spring Harbor Laboratory

Date: 15-03-2021

DOI: 10.1101/2021.03.14.435332

Abstract: Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project – Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N - and O -glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved “high coverage” and “high accuracy” glycoproteomics search strategies. This study concludes that erse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

Studies on the “Insoluble” Glycoprotein Complex from Human Colon

Publisher: Elsevier BV

Date: 05-1999

DOI: 10.1074/JBC.274.22.15828

Glycoprotein detection of 2-D separated proteins

Publisher: Humana Press

Date: 1999

DOI: 10.1385/1-59259-584-7:341

Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated and correlates with expression of bisecting GlcNAc-modified proteins

Publisher: American Association for Cancer Research (AACR)

Date: 15-08-2015

DOI: 10.1158/1557-3265.OVCASYMP14-POSTER-BIOL-1323

Abstract: Changes in the glycosylation of membrane proteins are a common feature of malignant transformation including ovarian cancer development. Of particular interest are N-linked glycans because these cell surface glycans mediate various cellular functions in cell-cell communication, cell adhesion, and cell motility. However the nature of these changes, their underlying molecular mechanisms, and the biological function of these glycans are poorly understood. In the present study, N-glycans were released from extracted membrane glycoproteins from ovarian cancer and normal ovarian epithelial cell lines and analysed using electrospray ionisation mass spectrometry RT-qPCR measured gene expression of related glycosyltransferases bisulfite sequencing analysed DNA methylation of the corresponding genes. We identified characteristic glycan features unique to membrane proteins of ovarian cancer cells. Among those ovarian cancer specific N-glycans were the ‘bisecting N-acetyl-glucosamine (GlcNAc)’ type. The bisecting GlcNAc is the product of the enzyme MGAT3 (beta-1,4-N-acetylglucosaminyltransferase 3) and the expression of the MGAT3 gene was elevated in ovarian cancer cell lines (SKOV3, IGROV1, A2780, OVCAR3) as compared to normal ovary surface epithelial (HOSE) cells. This elevated MGAT3 expression in ovarian cancer cells correlated with DNA hypomethylation at the transcription start site (TSS) of the MGAT3 promotor, which contains a large CpG island with 204 CpG dinucleotides located -600/+800bp relative to the TSS. This finding suggests that MGAT3 expression is epigenetically regulated. This was validated by the finding that the treatment of HOSE cells with the DNA-methyltransferase inhibitor 5-Aza increased MGAT3 expression and corresponded to a reduction in DNA methylation at the TSS. Taken together, we conclude that the elevated MGAT3 expression in ovarian cancer cells correlates with the presence of the resultant specific N-glycan substructures and with the epigenetic regulation of the associated enzymes by DNA methylation. These findings may stimulate the development of novel anti-glycan targeted drugs and clinical diagnostic tools for ovarian cancer Citation Format: Francis Jacob, Merrina Anugraham, Reto Kohler, Sheri Nixdorf, Arun Vijay Everest-Dass, André Fedier, Viola Heinzelmann-Schwarz, Nicolle H. Packer. Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated and correlates with expression of bisecting GlcNAc-modified proteins [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium Sep 8-9, 2014 Seattle, WA. Philadelphia (PA): AACR Clin Cancer Res 2015 (16 Suppl):Abstract nr POSTER-BIOL-1323.

Natural anti-glycan IgM recognize P1 glycosphingolipid expressed on ovarian cancer cells

Publisher: American Association for Cancer Research (AACR)

Date: 15-08-2015

DOI: 10.1158/1557-3265.OVCASYMP14-POSTER-BIOL-1322

Abstract: Recent research strongly suggests a role of plasma-derived anti-P1 antibodies (AGA) in ovarian cancer, as demonstrated by three independent glycan-based immunoassays. The level of these antibodies was lower in ovarian cancer patients and therefore discriminate cancer patients from healthy women. Here we investigate in a separate Australian cohort (n=155) whether IgM or IgG to P1 accounts for this discrimination and whether plasma matched ascites s les contain AGA. We also aimed to identify the P1 antigen in cancer tissue and cultured cells that are recognized by naturally occurring anti-P1 IgM and to investigate the potential function in respect to cell migration. Our results demonstrated that the IgM to P1 discriminates patients with ovarian cancer from healthy controls (p=0.0002) and that lower anti-P1 antibody levels are associated with a slightly higher risk for early relapse. Mass spectrometry identified P1 and structurally related epitopes in fresh tissue specimens and cultured cells. Ovarian cancer cell line IGROV1 was identified to be P1-positive while others (n=7) including human ovarian surface epithelial cells were negative determined by comprehensive flow cytometry. Epitope-mapped and characterized affinity purified anti-P1 antibodies from ascites were shown to bind P1-expressing cancer cells. IGROV1 was cell-sorted into two subpopulations, P1–high (66.1%) and P1-low (33.3%) and we found a significantly higher migration rate in P1-high expressing cells. Plasma-and ascites-derived natural anti-P1 IgM bind to the corresponding selfantigen expressed on the ovarian cancer cell surface. These findings are in concordance with the literature on natural IgM as part of the innate immune system recognizing carbo-neo-epitopes. These results deliver the first evidence that P1 may also be involved in cell migration and therefore may have a role in metastasis. Citation Format: Francis Jacob, Merrina Anugraham, Tatiana Pochechueva, Brian Wan-Chi Tse, Shahidul Alam, Rea Guertler, Nicolai V. Bovin, André Fedier, Neville F. Hacker, Margaret E. Huflejt, Nicolle Packer, Viola A. Heinzelmann-Schwarz. Natural anti-glycan IgM recognize P1 glycosphingolipid expressed on ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium Sep 8-9, 2014 Seattle, WA. Philadelphia (PA): AACR Clin Cancer Res 2015 (16 Suppl):Abstract nr POSTER-BIOL-1322.

Assessing the activity of antibodies conjugated to upconversion nanoparticles for immunolabeling

Publisher: Elsevier BV

Date: 05-0005

DOI: 10.1016/J.ACA.2022.339863

Abstract: Surface modification and functionalization is typically required to engineer upconversion nanoparticles (UCNPs) for biosensing and bioimaging applications. Nevertheless, despite various antibody conjugation methods having been applied to UCNPs, no consensus has been reached on the best choice, as the results from in idual studies are largely unable to be compared due to inadequate assessment of the properties of the conjugated products. Here, we introduce a systematic approach to quantitatively evaluate the biological activity of antibody-conjugated UCNPs. We determine that the optimal antibody conjugation efficiency to our colominic acid polysaccharide-coated UCNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS) coupling is approximately 70%, corresponding to 16 antibodies per nanoparticle of 63 nm hydrodynamic diameter, with on average 12 of the 16 antibodies maintaining their affinity to the target antigens. The binding ability of the antibody-conjugated UCNPs to the antigen was well preserved, as verified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and cellular imaging. This is the first study to quantitate the active antibody binding capacity of polysaccharide coated UCNP nanoparticles, offering a practical guideline for benchmarking functionalised UCNPs in future studies.

Structural analysis of glycoprotein sialylation – Part I: pre-LC-MS analytical strategies

Publisher: Royal Society of Chemistry (RSC)

Date: 2013

DOI: 10.1039/C3RA42960A

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster

Publisher: American Society for Microbiology

Date: 07-1994

DOI: 10.1128/JB.176.13.4144-4156.1994

Abstract: Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -- )-beta-D-Galf-(1-- )-alpha-D-Glcp- (1-- )-alpha-L-Rhap-(1-- )-alpha-D-GlcpNAc-(1-- , with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.

Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions*.

Publisher: Elsevier BV

Date: 08-2017

DOI: 10.1074/MCP.M116.066746

The glycosphingolipid P-1 is an ovarian cancer-associated carbohydrate antigen involved in migration

Publisher: Springer Science and Business Media LLC

Date: 28-08-2014

DOI: 10.1038/BJC.2014.455

N-Glycans Modulate the Function of Human Corticosteroid-Binding Globulin

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1074/MCP.M111.009100

Characterization and downstream mannose phosphorylation of human recombinant α‐L‐iduronidase produced in Arabidopsis complex glycan‐deficient (cgl) seeds

Publisher: Wiley

Date: 31-07-2013

DOI: 10.1111/PBI.12096

Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D2NA00036A

Abstract: Nanodiamonds were coated in lectins to target glycan receptors on astrocytes, neurons and microglia. The uptake in each cell type was variable depending on their coating of Aleuria aurantia lectin, wheat germ agglutinin or tomato lectin.

Emerging roles of protein mannosylation in inflammation and infection

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1016/J.MAM.2016.04.004

Abstract: Proteins are frequently modified by complex carbohydrates (glycans) that play central roles in maintaining the structural and functional integrity of cells and tissues in humans and lower organisms. Mannose forms an essential building block of protein glycosylation, and its functional involvement as components of larger and erse α-mannosidic glycoepitopes in important intra- and intercellular glycoimmunological processes is gaining recognition. With a focus on the mannose-rich asparagine (N-linked) glycosylation type, this review summarises the increasing volume of literature covering human and non-human protein mannosylation, including their structures, biosynthesis and spatiotemporal expression. The review also covers their known interactions with specialised host and microbial mannose-recognising C-type lectin receptors (mrCLRs) and antibodies (mrAbs) during inflammation and pathogen infection. Advances in molecular mapping technologies have recently revealed novel immuno-centric mannose-terminating truncated N-glycans, termed paucimannosylation, on human proteins. The cellular presentation of α-mannosidic glycoepitopes on N-glycoproteins appears tightly regulated α-mannose determinants are relative rare glycoepitopes in physiological extracellular environments, but may be actively secreted or leaked from cells to transmit potent signals when required. Simultaneously, our understanding of the molecular basis on the recognition of mannosidic epitopes by mrCLRs including DC-SIGN, mannose receptor, mannose binding lectin and mrAb is rapidly advancing, together with the functional implications of these interactions in facilitating an effective immune response during physiological and pathophysiological conditions. Ultimately, deciphering these complex mannose-based receptor-ligand interactions at the detailed molecular level will significantly advance our understanding of immunological disorders and infectious diseases, promoting the development of future therapeutics to improve patient clinical outcomes.

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. II. Proton and carbon magnetic resonance spectra

Publisher: Elsevier BV

Date: 12-1985

DOI: 10.1016/0005-2736(85)90088-4

Tandem mass spectra of glycan substructures enable the multistage mass spectrometric identification of determinants on oligosaccharides

Publisher: Wiley

Date: 16-04-2013

DOI: 10.1002/RCM.6527

Abstract: Glycosylation of proteins and lipids affects many biological processes, such as host-pathogen interactions, cell communication, and initiation of the immune responses. Terminal glycan substructures, or determinants, often govern the function or recognition of the carrier glycoconjugate and modulate these processes. In this study we describe a strategy using multistage mass spectrometry to identify and confirm these glycan substructures. An online tandem mass spectrometry (MS(2)) spectral fragment library of glycan substructures that typically occur at the non-reducing terminus of glycoconjugates was created to enable the easier identification and confirmation of glycan determinants on oligosaccharides released from glycoproteins. Oligosaccharides were separated by porous graphitized carbon capillary chromatography and analysed by ion trap MS. Candidate product ions that constitute the glycan substructure mass were identified in the MS(2) product ion spectrum, and used as the precursor ion for subsequent MS(3) fragmentation. The resulting MS(3) spectrum was matched against the MS(2) spectral fragment library to identify the glycan substructure(s) that comprise the parent oligosaccharide. Thirty biologically important terminal glycan determinants commonly observed on glycoconjugates were fragmented by positive and negative ion mass spectrometry and the MS(2) product ion masses manually annotated and stored in the UniCarb-DB online database. Negative ion tandem mass spectra were especially useful in assigning isobaric glycan structures. We have applied this strategy for the identification of the sulphation, blood group antigens and sialic acid linkages on complex N-and O-glycans released from glycoproteins. We show the potential of these glycan substructure MS(2) spectra in the negative ionization mode to facilitate the assignment of determinants on N- and O-linked glycans released from glycoproteins. Comparing the structural feature ions of known glycan reference substructures assists in the annotation of complex glycan product ion spectra, and can remove the need for other orthogonal confirmation analyses such as sequential glycosidase digestion.

Fiber Supplements Derived From Sugarcane Stem, Wheat Dextrin and Psyllium Husk Have Different In Vitro Effects on the Human Gut Microbiota

Publisher: Frontiers Media SA

Date: 20-07-2018

DOI: 10.3389/FMICB.2018.01618

Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSⁿ.

Publisher: American Chemical Society (ACS)

Date: 05-2004

DOI: 10.1016/J.JASMS.2004.01.002

Towards a standardized bioinformatics infrastructure for N- and O-glycomics

Publisher: Springer Science and Business Media LLC

Date: 22-07-2019

DOI: 10.1038/S41467-019-11131-X

Abstract: The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N - and O -linked glycans ( N - and O -glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.

UniCarb-DB: a database resource for glycomic discovery

Publisher: Oxford University Press (OUP)

Date: 12-03-2011

DOI: 10.1093/BIOINFORMATICS/BTR137

Abstract: Summary: Glycosylation is one of the most important post-translational modifications of proteins, known to be involved in pathogen recognition, innate immune response and protection of epithelial membranes. However, when compared to the tools and databases available for the processing of high-throughput proteomic data, the glycomic domain is severely lacking. While tools to assist the analysis of mass spectrometry (MS) and HPLC are continuously improving, there are few resources available to support liquid chromatography (LC)–MS/MS techniques for glycan structure profiling. Here, we present a platform for presenting oligosaccharide structures and fragment data characterized by LC–MS/MS strategies. The database is annotated with high-quality datasets and is designed to extend and reinforce those standards and ontologies developed by existing glycomics databases. Availability: www.unicarb-db.org Contact: matthew.c bell@mq.edu.au

Protein glycosylation pathways in filamentous fungi

Publisher: Oxford University Press (OUP)

Date: 26-05-2008

DOI: 10.1093/GLYCOB/CWN044

Abstract: Glycosylation of proteins is important for protein stability, secretion, and localization. In this study, we have investigated the glycan synthesis pathways of 12 filamentous fungi including those of medical/agricultural/industrial importance for which genomes have been recently sequenced. We have adopted a systems biology approach to combine the results from comparative genomics techniques with high confidence information on the enzymes and fungal glycan structures, reported in the literature. From this, we have developed a composite representation of the glycan synthesis pathways in filamentous fungi (both N- and O-linked). The N-glycosylation pathway in the cytoplasm and endoplasmic reticulum was found to be highly conserved evolutionarily across all the filamentous fungi considered in the study. In the final stages of N-glycan synthesis in the Golgi, filamentous fungi follow the high mannose pathway as in Saccharomyces cerevisiae, but the level of glycan mannosylation is reduced. Highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures have also been identified in the filamentous fungi. O-Linked glycosylation in filamentous fungi was seen to be highly conserved with many mannosyltransferases that are similar to those in S. cerevisiae. However, highly variable and erse O-linked glycans also exist. We have developed a web resource for presenting the compiled data with user-friendly query options, which can be accessed at www.fungalglycans.org. This resource can assist attempts to remodel glycosylation of recombinant proteins expressed in filamentous fungal hosts.

Enhancing structural characterisation of glucuronidated O-linked glycans using negative mode ion trap higher energy collision-induced dissociation mass spectrometry.

Publisher: Wiley

Date: 18-04-2017

DOI: 10.1002/RCM.7851

Abstract: High protein production and secretion with eukaryotic glycosylation machinery make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The N-glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients but O-glycosylation has been less extensively studied. O-Glycans and glycopeptides from secreted proteins were separated by porous graphitised carbon and C-18 liquid chromatography, respectively. O-Glycans were analysed in negative ion mode by electrospray ionisation linear ion trap mass spectrometry and glycopeptides in positive ion mode by electrospray ionisation hybrid quadrupole-orbitrap mass spectrometry. Tandem mass spectrometry was used on O-glycans and glycopeptides including ion trap higher energy collision-induced dissociation (tHCD) to detect glycan fragments not detectable with standard ion trap fragmentation. tHCD allowed targeted MS Negative mode ion trap higher energy collision-induced dissociation allowed detection and targeted MS These are the first reported O-glycans to contain acidic sugars in fungi and they could have significant implications for cellobiohydrolase I structure and activity as well as the activity of recombinant proteins expressed in this host system. Copyright © 2017 John Wiley & Sons, Ltd.

Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

Publisher: Royal Society of Chemistry (RSC)

Date: 2019

DOI: 10.1039/C9AN00486F

Abstract: Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins.

Glycomics@ExPASy: Bridging the Gap

Publisher: Elsevier BV

Date: 11-2018

DOI: 10.1074/MCP.RA118.000799

The relationship between the structures of the O polysaccharides from Escherichia coli O17 and O16

Publisher: Elsevier BV

Date: 09-1997

DOI: 10.1016/S0008-6215(97)00177-8

Abstract: The chemical structure of the O16 antigen from the lipopolysaccharide of Escherichia coli strain P4 has been determined. Comparison with the structures of other O16 antigens and that of the O17 antigen explains the previously reported cross-reaction of O antigen from the O16 strain K-12 with anti-O17 antibody [D. Liu and P.R. Reeves, Microbiology, 140 (1994) 49-57].

Reply by the authors to ‘New urinary EPO drug testing method using two-dimensional gel electrophoresis’

Publisher: Elsevier BV

Date: 11-2006

DOI: 10.1016/J.CCA.2006.03.022

Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells

Publisher: Wiley

Date: 29-06-2019

DOI: 10.1096/FJ.201900559R

SugarBindDB, a resource of glycan-mediated host–pathogen interactions

Publisher: Oxford University Press (OUP)

Date: 17-11-2015

DOI: 10.1093/NAR/GKV1247

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.

Publisher: Elsevier BV

Date: 07-2002

DOI: 10.1074/MCP.M200020-MCP200

Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

Publisher: American Chemical Society (ACS)

Date: 20-04-2013

DOI: 10.1007/S13361-013-0610-4

Abstract: Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure-function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological s le. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

Author Correction: Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis

Publisher: Springer Science and Business Media LLC

Date: 10-12-2021

DOI: 10.1038/S41592-021-01368-0

Characterisation of the slime gland secretion from the peripatus, Euperipatoides kanangrensis (Onychophora: Peripatopsidae)

Publisher: Elsevier BV

Date: 12-1999

DOI: 10.1016/S0305-0491(99)00145-5

Identification and quantitation of cysteine in proteins separated by gel electrophoresis

Publisher: Elsevier BV

Date: 07-1998

DOI: 10.1016/S0021-9673(98)00319-7

Abstract: A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 80 degrees C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 37 degrees C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

The GlySpace Alliance: toward a collaborative global glycoinformatics community

Publisher: Oxford University Press (OUP)

Date: 10-2019

DOI: 10.1093/GLYCOB/CWZ078

Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012

Publisher: Elsevier BV

Date: 10-2013

DOI: 10.1074/MCP.M113.030643

Glycoproteome remodelling in MLL-rearranged B-cell precursor acute lymphoblastic leukemia

Publisher: Cold Spring Harbor Laboratory

Date: 30-06-2021

DOI: 10.1101/2021.06.25.21259296

Abstract: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are urgently needed. Currently no multi omics data set for primary MLL r patient cells exists that integrates transcriptomics, proteomics and glycomics to gain an inclusive picture of theranostic targets. We have integrated transcriptomics, proteomics and glycomics to i) obtain the first inclusive picture of primary patient BCP-ALL cells and identify molecular signatures that distinguish leukemic from normal precursor B-cells and ii) better understand the benefits and limitations of the applied technologies to deliver deep molecular sequence data across major cellular biopolymers. MLL-r cells feature an extensive remodelling of their glycocalyx, with increased levels of Core 2-type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation. Notably, glycosaminoglycan remodelling from chondroitin sulfate to heparan sulfate was observed. A survival screen, to determine if glycan remodelling enzymes are redundant, identified MGAT1 and NGLY1, essential components of the N-glycosylation/degradation pathway, as highly relevant within this in vitro screening. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. While there is overall good correlation between transcriptomics and proteomics data, we demonstrate that a systematic combined multi- omics approach delivers important diagnostic information that is missed when applying a single omics technology. Apart from confirming well-known MLL-r BCP-ALL glycoprotein markers, our integrated multi-omics workflow discovered previously unidentified diagnostic/therapeutic protein targets.

Biosimilarity and Interchangeability: Principles and Evidence: A Systematic Review

Publisher: Springer Science and Business Media LLC

Date: 17-01-2018

DOI: 10.1007/S40259-017-0256-Z

Stable Upconversion Nanohybrid Particles for Specific Prostate Cancer Cell Immunodetection

Publisher: Springer Science and Business Media LLC

Date: 22-11-2016

DOI: 10.1038/SREP37533

Abstract: Prostate cancer is one of the male killing diseases and early detection of prostate cancer is the key for better treatment and lower cost. However, the number of prostate cancer cells is low at the early stage, so it is very challenging to detect. In this study, we successfully designed and developed upconversion immune-nanohybrids (UINBs) with sustainable stability in a physiological environment, stable optical properties and highly specific targeting capability for early-stage prostate cancer cell detection. The developed UINBs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and luminescence spectroscopy. The targeting function of the biotinylated antibody nanohybrids were confirmed by immunofluorescence assay and western blot analysis. The UINB system is able to specifically detect prostate cancer cells with stable and background-free luminescent signals for highly sensitive prostate cancer cell detection. This work demonstrates a versatile strategy to develop UCNPs based sustainably stable UINBs for sensitive diseased cell detection.

Comprehensive glycomics comparison between colon cancer cell cultures and tumours: Implications for biomarker studies

Publisher: Elsevier BV

Date: 05-0004

DOI: 10.1016/J.JPROT.2014.05.002

Abstract: Altered glycosylation is commonly observed in colorectal cancer. In vitro models are frequently used to study this cancer but little is known about the differences that may exist between these model cell systems and tumour tissue. We have compared the membrane protein glycosylation of five colorectal cancer cell lines (SW1116, SW480, SW620, SW837, LS174T) with epithelial cells from colorectal tumours using liquid chromatography tandem mass spectrometry. Remarkably, there were five abundant O-glycans in the tumour cells that were undetected in the low-mucin producing cell lines, although two were found in the mucinous LS174T cells. The O-glycans included the well-known glycan cancer marker, sialyl-Tn, which has been associated with mucins. Using qRT-PCR, sialyl-Tn expression was found to be associated with an increase in α2,6-sialyltransferase gene (ST6GALNAC1) and a decrease in core 1 synthase gene (C1GALT1) in LS174T cells. The expression of a subset of mucins (MUC2, MUC6, MUC5B) was also correlated with sialyl-Tn expression in LS174T cells. Overall, the membrane protein glycosylation of the model cell lines was found to differ from each other and from the epithelial cells of tumour tissue. These findings should be noted in the design of biomarker discovery experiments particularly when cell surface targets are being investigated. The extent of protein glycosylation differences between in vitro cell lines and ex vivo tumours in colorectal cancer research is unknown. Our study expands current knowledge by characterising the membrane protein glycosylation profiles of five different colorectal cancer cell lines and of epithelial cells derived from resected colorectal cancer tumour tissue, using liquid chromatography tandem mass spectrometry. The detailed structural differences found in both N- and O-linked glycan structures on the membrane glycoproteins were determined and correlated with the mRNA expression of the relevant proteins in the cell lines. The glycosylation differences found between cultured cancer cell lines and epithelial cells from tumour tissue have important implications for glycan biomarker discovery.

FUT1genetic variants impact protein glycosylation of porcine intestinal mucosa

Publisher: Oxford University Press (OUP)

Date: 07-02-2016

DOI: 10.1093/GLYCOB/CWW009

Abstract: A massive use of antibiotics in industrial pig production is a major cause of the rapidly rising bacterial resistance to antibiotics. An enhanced understanding of infectious diseases and of host-microbe interactions has the potential to explore alternative ways to improve pig health and reduce the need for antibiotics. Host-microbe interactions depend on host-expressed glycans and microbe-carrying lectins. In this study, a G > A (nucleotide 307) missense mutation in the porcine α1,2fucosyltransferase 1 gene (FUT1), which has been reported to prevent infections by the common porcine enteric pathogen F18 fimbriated Escherichia coli, provided a unique opportunity to study glycan structures potentially involved in intestinal infections. N- and O-Linked glycans of the intestinal mucosa proteins were characterized in detail using LC-MS/MS. Relative abundances of all glycans were determined and compared between four heterozygous pigs (FUT1-307(A/G)) and four age-matched homozygous pigs from the same 2 litters carrying the missense FUT1 gene constellation (FUT1-307(A/A)). None of the characterized 48 N-linked glycans was found to be regulated by the FUT1 missense mutation, while 11 of the O-linked glycans showed significantly altered abundances between the two genotypes. The overall abundance of H-antigen carrying structures was decreased fivefold, while H-antigen precursors and sialylated structures were relatively more abundant in pigs with the FUT1 missense mutation. These results provide insight into the role of FUT1 on intestinal glycosylation, improve our understanding of how variation in FUT1 can modulate host-microbe interactions, and suggest that the FUT1 genetic variant may help to improve pig gut health.

Concurrent Sessions

Publisher: Wiley

Date: 07-2015

DOI: 10.1111/FEBS.13320

Localization of O-glycosylation sites on glycopeptide fragments from lactation-associated MUC1. All putative sites within the tandem repeat are glycosylation targets in vivo

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1074/JBC.272.40.24780

High sample throughput phosphoamino acid analysis of proteins separated by one- and two-dimensional gel electrophoresis

Publisher: Elsevier BV

Date: 03-1997

DOI: 10.1016/S0021-9673(96)00931-4

Site-SpecificN-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

Publisher: American Chemical Society (ACS)

Date: 04-2016

DOI: 10.1007/S13361-016-1378-0

Abstract: Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling. Graphical Abstract ᅟ.

Analysis of O-Linked Reducing Oligosaccharides Released by an In-line Flow System

Publisher: Elsevier BV

Date: 06-2002

DOI: 10.1006/ABIO.2002.5657

Human disease glycomics: technology advances enabling protein glycosylation analysis – part 2

Publisher: Informa UK Limited

Date: 20-03-2018

DOI: 10.1080/14789450.2018.1448710

Abstract: The changes in glycan structures have been attributed to disease states for several decades. The surface glycosylation pattern is a signature of physiological state of a cell. In this review we provide a link between observed substructural glycan changes and a range of diseases. Areas covered: We highlight biologically relevant glycan substructure expression in cancer, inflammation, neuronal diseases and diabetes. Furthermore, the alterations in antibody glycosylation in a disease context are described. Expert commentary: Advances in technologies, as described in Part 1 of this review have now enabled the characterization of specific glycan structural markers of a range of disease states. The requirement of including glycomics in cross-disciplinary omics studies, such as genomics, proteomics, epigenomics, transcriptomics and metabolomics towards a systems glycobiology approach to understanding disease mechanisms and management are highlighted.

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines

Publisher: Springer Science and Business Media LLC

Date: 28-10-2015

DOI: 10.1007/S10719-015-9625-3

Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions

Publisher: Portland Press Ltd.

Date: 26-04-2005

DOI: 10.1042/BJ20041641

Abstract: SMG (submucosal gland) secretions are a major component of the airway surface liquid, are associated with innate immunity in the lung, and have been reported to be altered in lung disease. Changes in lung mucosal glycosylation have been reported in CF (cystic fibrosis), which may be responsible for differential bacterial binding to glycosylated components in the lung mucosa and hence increased pre-disposition to pulmonary infection. Glycoproteomic analysis was performed on SMG secretions collected from explanted bronchial tissue of subjects with severe lung disease, with and without CF, and controls without lung disease. Mucins MUC5B and MUC5AC were shown to be the dominant high-molecular-mass glycoprotein components, with a minor non-mucin glycoprotein component, gp-340, also present. Oligosaccharides containing blood-group determinants corresponding to subjects' blood type were abundant on MUC5B/MUC5AC, as were Lewis-type epitopes and their sialylated analogues, which are ligands for pathogens and leucocytes. No significant differences were found in the glycosylation of MUC5B/MUC5AC or gp-340 between CF and non-CF subjects with severe lung disease, implying that CF does not influence SMG secretion mucin glycosylation in end-stage lung disease. There were also no significant differences found in the glycosylation of these components in severe lung disease compared with non-diseased lungs. This suggests that previously reported changes in the glycosylation of respiratory glycoconjugates in CF, and other pulmonary conditions, are not due to the glycosylation of components in SMG secretions, but may involve other secretions, responses or extracellular factors.

The biochemical nature of the cell envelope of a high cellulase-secreting mutant differs from that of the Trichoderma reesei wild type

Publisher: Elsevier BV

Date: 08-1995

DOI: 10.1016/0168-1656(95)00064-W

Sialic acid plays a pivotal role in licensing Citrobacter rodentium’s transition from the intestinal lumen to a mucosal adherent niche

Publisher: Proceedings of the National Academy of Sciences

Date: 03-07-2023

DOI: 10.1073/PNAS.2301115120

Abstract: Enteric bacterial pathogens pose significant threats to human health however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses and an established microbiota remain poorly defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium , its virulence strategy likely involves metabolic adaptation to the host’s intestinal luminal environment, as a necessary precursor to reach and infect the mucosal surface. Suspecting this adaptation involved the intestinal mucus layer, we found that C. rodentium was able to catabolize sialic acid, a monosaccharide derived from mucins, and utilize it as its sole carbon source for growth. Moreover, C. rodentium also sensed and displayed chemotactic activity toward sialic acid. These activities were abolished when the nanT gene, encoding a sialic acid transporter, was deleted (Δ nanT ). Correspondingly, the Δ nanT C. rodentium strain was significantly impaired in its ability to colonize the murine intestine. Intriguingly, sialic acid was also found to induce the secretion of two autotransporter proteins, Pic and EspC, which possess mucinolytic and host-adherent properties. As a result, sialic acid enhanced the ability of C. rodentium to degrade intestinal mucus (through Pic), as well as to adhere to intestinal epithelial cells (through EspC). We thus demonstrate that sialic acid, a monosaccharide constituent of the intestinal mucus layer, functions as an important nutrient and a key signal for an A/E bacterial pathogen to escape the colonic lumen and directly infect its host’s intestinal mucosa.

Program and Abstracts for 2016 Annual Meeting of the Society for Glycobiology

Publisher: Oxford University Press (OUP)

Date: 31-10-2016

DOI: 10.1093/GLYCOB/CWW110

Chloroplast development and the synthesis of chlorophyll and protochlorophyllide in Zostera transferred to darkness

Publisher: Springer Science and Business Media LLC

Date: 1985

DOI: 10.1007/BF00398091

Detection of glycoproteins in gels and blots

Publisher: Humana Press

Date: 2002

DOI: 10.1385/1-59259-169-8:761

Influence of surface chemistry on the formation of a protein corona on nanodiamonds

Publisher: Royal Society of Chemistry (RSC)

Date: 2019

DOI: 10.1039/C9TB00445A

Abstract: The protein corona of nanodiamonds is dominated by low molecular weight proteins and is largely independent of surface chemistry. The pre-incubation of nanodiamonds in serum and the formation of a protein corona decrease the production of reactive oxygen species, increasing the cell viability of macrophages.

Phenotypic profiling of pancreatic ductal adenocarcinoma plasma-derived small extracellular vesicles for cancer diagnosis and cancer stage prediction: a proof-of-concept study

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D2AY00536K

Abstract: Phenotype profiling of plasma-derived sEVs using SERS based assay for PDAC diagnosis and cancer stage prediction.

Blood group antigen expression is involved in C. albicans interaction with buccal epithelial cells

Publisher: Springer Science and Business Media LLC

Date: 17-09-2017

DOI: 10.1007/S10719-016-9726-7

Abstract: Human blood group polymorphisms are known to be determined by the expression of A, B or H antigens and the Lewis antigens. Protection against microbial infections has been associated with inheritance of polymorphisms in genes encoding and regulating the expression of ABH and Lewis antigens in bodily secretions and epithelial tissue surfaces, subsequently resulting in the presentation of different glycosylated terminal antigens on the cell surface. We investigated the role of blood group antigens in ersifying the glycosylation of buccal epithelial cells (BEC) that line the oral cavity. Specifically, we characterized and statistically evaluated the expression of histo-blood group (A, B, O) antigens on N-and O-linked glycans from BEC membrane proteins of various in iduals that represented different blood group type and secretor status using a porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry (PGC-LC-ESI-MS) based glycomics approach. From these BEC membrane proteins a total of 77 N-glycan and 96 O-glycan structures were structurally characterized from 19 in iduals and relatively quantitated. The N-glycans from the secretor in iduals did not express any A/B blood group determinants, but contained several terminal H-antigens. Apart from the non-secretors, the N-glycan profiles of BEC from all blood groups displayed similar glycan types, while varying in their relative intensities between in iduals. However, multivariate analysis of the O-glycans from in iduals displayed segregation patterns clearly associated with their blood group type and secretor status. In adhesion assays the oral pathogen Candida albicans showed a significantly higher interaction to blood group O type BECs relative to other blood groups.

Influence of an ER-retention signal on the N-glycosylation of recombinant human α-l-iduronidase generated in seeds of Arabidopsis

Publisher: Springer Science and Business Media LLC

Date: 23-03-2012

DOI: 10.1007/S11103-012-9902-5

Abstract: Processes associated with late events of N-glycosylation within the plant Golgi complex are a major limitation to the use of plant-based systems to produce recombinant pharmaceutical proteins for parenteral administration. Specifically, sugars added to the N-glycans of a recombinant protein during glycan maturation to complex forms (e.g. β1,2 xylose and α1,3 fucose) can render the product immunogenic. In order to avoid these sugars, the human enzyme α-L-iduronidase (IDUA, EC 3.2.1.76), with a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of complex-glycan-deficient (cgl) mutant and wild-type (Col-0) Arabidopsis thaliana, under the control of regulatory (5'-, signal-peptide-encoding-, and 3'-) sequences from the arcelin 5-I gene of Phaseolus vulgaris (cgl-IDUA-SEKDEL and Col-IDUA-SEKDEL, respectively). The SEKDEL motif had no adverse effect on the specific activity of the purified enzyme. Surprisingly, the majority of the N-glycans of Col-IDUA-SEKDEL were complex N-glycans (i.e. contained xylose and/or fucose) (88 %), whereas complex N-glycans comprised a much lower proportion of the N-glycans of cgl-IDUA-SEKDEL (26 %), in which high-mannose forms were predominant. In contrast to the non-chimeric IDUA of cgl seeds, which is mainly secreted into the extracellular spaces, the addition of the SEKDEL sequence to human recombinant IDUA expressed in the same background led to retention of the protein in ER-derived vesicles/compartments and its partial localization in protein storage vacuoles. Our data support the contention that the use of a C-terminal ER retention motif as an effective strategy to prevent or reduce complex N-glycan formation, is protein specific.

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. I. Phosphorus magnetic resonance spectra

Publisher: Elsevier BV

Date: 12-1985

DOI: 10.1016/0005-2736(85)90087-2

The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data

Publisher: Oxford University Press (OUP)

Date: 22-11-2016

DOI: 10.1093/GLYCOB/CWW118

Proteomic Analysis of the Genetic Premature Aging Disease Hutchinson Gilford Progeria Syndrome Reveals Differential Protein Expression and Glycosylation

Publisher: American Chemical Society (ACS)

Date: 28-08-2003

DOI: 10.1021/PR034035K

Abstract: Proteomics has revealed differential protein expression and glycosylation in membrane proteins from premature aging Hutchinson-Gilford progeria syndrome fibroblasts (progeria). Progeria is a rare autosomal dominant genetic disorder of premature aging characterized by marked growth retardation and specific, progressive, premature senescent changes of the skin and other tissues. Affected children live to an average age of 13 years. The 1q20-24 region of chromosome 1 which codes for one of these proteins, lamin A/C, has previously been implicated by Brown et al. (1990) who described identical twins with progeria, where cytogenetic analysis showed an inverted insertion in the long arm of the chromosome in 70% of cells. Luengo et al. (2002) similarly reported an interstitial deletion of chromosome 1q23, in a 9-year-old patient with a classic clinical picture of progeria.

Engineered short forms of perlecan enhance angiogenesis by potentiating growth factor signalling

Publisher: Elsevier BV

Date: 10-2023

DOI: 10.1016/J.JCONREL.2023.08.052

Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation

Publisher: Springer Science and Business Media LLC

Date: 06-1994

DOI: 10.1007/BF00731216

GlycoBioinformatics

Publisher: Beilstein Institut

Date: 09-11-2021

DOI: 10.3762/BJOC.17.184

Pseudomonas aeruginosa Cell Membrane Protein Expression from Phenotypically Diverse Cystic Fibrosis Isolates Demonstrates Host-Specific Adaptations

Publisher: American Chemical Society (ACS)

Date: 09-06-2016

DOI: 10.1021/ACS.JPROTEOME.6B00058

Abstract: Pseudomonas aeruginosa is a Gram-negative, nosocomial, highly adaptable opportunistic pathogen especially prevalent in immuno-compromised cystic fibrosis (CF) patients. The bacterial cell surface proteins are important contributors to virulence, yet the membrane subproteomes of phenotypically erse P. aeruginosa strains are poorly characterized. We carried out mass spectrometry (MS)-based proteome analysis of the membrane proteins of three novel P. aeruginosa strains isolated from the sputum of CF patients and compared protein expression to the widely used laboratory strain, PAO1. Microbes were grown in planktonic growth condition using minimal M9 media, and a defined synthetic lung nutrient mimicking medium (SCFM) limited passaging. Two-dimensional LC-MS/MS using iTRAQ labeling enabled quantitative comparisons among 3171 and 2442 proteins from the minimal M9 medium and in the SCFM, respectively. The CF isolates showed marked differences in membrane protein expression in comparison with PAO1 including up-regulation of drug resistance proteins (MexY, MexB, MexC) and down-regulation of chemotaxis and aerotaxis proteins (PA1561, PctA, PctB) and motility and adhesion proteins (FliK, FlgE, FliD, PilJ). Phenotypic analysis using adhesion, motility, and drug susceptibility assays confirmed the proteomics findings. These results provide evidence of host-specific microevolution of P. aeruginosa in the CF lung and shed light on the adaptation strategies used by CF pathogens.

Comprehensive analysis of the N-glycan biosynthetic pathway using bioinformatics to generate UniCorn: A theoretical N-glycan structure database

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.CARRES.2016.05.012

Abstract: Glycan structures attached to proteins are comprised of erse monosaccharide sequences and linkages that are produced from precursor nucleotide-sugars by a series of glycosyltransferases. Databases of these structures are an essential resource for the interpretation of analytical data and the development of bioinformatics tools. However, with no template to predict what structures are possible the human glycan structure databases are incomplete and rely heavily on the curation of published, experimentally determined, glycan structure data. In this work, a library of 45 human glycosyltransferases was used to generate a theoretical database of N-glycan structures comprised of 15 or less monosaccharide residues. Enzyme specificities were sourced from major online databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan, Consortium for Functional Glycomics (CFG), Carbohydrate-Active enZymes (CAZy), GlycoGene DataBase (GGDB) and BRENDA. Based on the known activities, more than 1.1 million theoretical structures and 4.7 million synthetic reactions were generated and stored in our database called UniCorn. Furthermore, we analyzed the differences between the predicted glycan structures in UniCorn and those contained in UniCarbKB (www.unicarbkb.org), a database which stores experimentally described glycan structures reported in the literature, and demonstrate that UniCorn can be used to aid in the assignment of ambiguous structures whilst also serving as a discovery database.

High-grade serous ovarian and peritoneal cancers display distinct genetic and post-translational signatures – a criterion to treat them differently?

Publisher: Georg Thieme Verlag KG

Date: 13-10-2016

DOI: 10.1055/S-0036-1593244

Blood group antigen recognition via the group a streptococcal m protein mediates host colonization

Publisher: American Society for Microbiology

Date: 08-03-2017

DOI: 10.1128/MBIO.02237-16

Abstract: Streptococcus pyogenes (group A streptococcus [GAS]) is responsible for over 500,000 deaths worldwide each year. The highly virulent M1T1 GAS clone is one of the most frequently isolated serotypes from streptococcal pharyngitis and invasive disease. The oral epithelial tract is a niche highly abundant in glycosylated structures, particularly those of the ABO(H) blood group antigen family. Using a high-throughput approach, we determined that a strain representative of the globally disseminated M1T1 GAS clone 5448 interacts with numerous, structurally erse glycans. Preeminent among GAS virulence factors is the surface-expressed M protein. M1 protein showed high affinity for several terminal galactose blood group antigen structures. Deletion mutagenesis shows that M1 protein mediates glycan binding via its B repeat domains. Association of M1T1 GAS with oral epithelial cells varied significantly as a result of phenotypic differences in blood group antigen expression, with significantly higher adherence to those cells expressing H antigen structures compared to cells expressing A, B, or AB antigen structures. These data suggest a novel mechanism for GAS attachment to host cells and propose a link between host blood group antigen expression and M1T1 GAS colonization. IMPORTANCE There has been a resurgence in group A streptococcal (GAS) invasive disease, which has been paralleled by the emergence of the highly virulent M1T1 GAS clone. Intensive research has focused on mechanisms that contribute to the invasive nature of this serotype, while the mechanisms that contribute to host susceptibility to disease and bacterial colonization and persistence are still poorly understood. The M1T1 GAS clone is frequently isolated from the throat, an environment highly abundant in blood group antigen structures. This work examined the interaction of the M1 protein, the preeminent GAS surface protein, against a wide range of host-expressed glycan structures. Our data suggest that susceptibility to infection by GAS in the oral tract may correlate with phenotypic differences in host blood group antigen expression. Thus, variations in host blood group antigen expression may serve as a selective pressure contributing to the dissemination and overrepresentation of M1T1 GAS.

Glycobiology and proteomics: Is mass spectrometry the holy grail?

Publisher: Wiley

Date: 08-1998

DOI: 10.1002/ELPS.1150191105

Abstract: One characteristic of glycoproteins is that they are separated by two-dimensional electrophoresis (2-D PAGE) into typical 'trains' of protein spots which separate on the basis of different isoelectric point (pI) and/or molecular mass. The pattern of these trains often varies in development and disease. While the isoforms differ both in the number of sites of glycosylation and the types of carbohydrate attached to the protein, classical methods of glycan analysis are insensitive at the levels typically separated by 2-D PAGE. Developments in mass spectrometry technologies have enabled the characterization of most of the oligosaccharide attributes to be determined on picomole amounts of protein. These techniques are beginning to allow the glycoform heterogeneity on 2-D separated glycoproteins to be analyzed.

An in vivo approach for the identification of acceptor sites for O- glycosyltransferases

Publisher: American Chemical Society (ACS)

Date: 04-1997

DOI: 10.1021/BI9617825

A general approach to desalting oligosaccharides released from glycoproteins

Publisher: Springer Science and Business Media LLC

Date: 1998

DOI: 10.1023/A:1006983125913

Abstract: Desalting of sugar s les is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for ex le, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and beta-eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.

Polysialic Acid Regulates Sympathetic Outflow by Facilitating Information Transfer within the Nucleus of the Solitary Tract

Publisher: Society for Neuroscience

Date: 02-06-2017

DOI: 10.1523/JNEUROSCI.0200-17.2017

Glycoconjugates in human milk

Publisher: Oxford University Press (OUP)

Date: 09-2013

DOI: 10.1093/GLYCOB/CWT072

Abstract: Breastfeeding is known to have many health benefits for a newborn. Not only does human milk provide an excellent source of nutrition, it also contains components that protect against infection from a wide range of pathogens. Some of the protective properties of human milk can be attributed to the immunoglobulins. Yet, there is another level of defense provided by the "sweet" protective agents that human milk contains, including free oligosaccharides, glycoproteins and glycolipids. Sugar epitopes in human milk are similar to the glycan receptors that serve as pathogen adhesion sites in the human gastrointestinal tract and other epithelial cell surfaces hence, the milk glycans can competitively bind to and remove the disease-causing microorganisms before they cause infection. The protective value of free oligosaccharides in human milk has been well researched and documented. Human milk glycoconjugates have received less attention but appear to play an equally important role. Here, we bring together the breadth of research that has focused on the protective mechanisms of human milk glycoconjugates, with a particular focus on the glycan moieties that may play a role in disease prevention. In addition, human milk glycoconjugates are compared with bovine milk glycoconjugates in terms of their health benefits for the human infant.

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates

Publisher: Springer Science and Business Media LLC

Date: 14-03-2018

DOI: 10.1038/S41598-018-22702-1

Abstract: Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

Long-term intrathecal administration of morphine vs. baclofen

Publisher: Oxford University Press (OUP)

Date: 09-09-2022

DOI: 10.1093/GLYCOB/CWAB098

Abstract: Opioid use for treatment of persistent pain has increased dramatically over the past two decades, but it has not resulted in improved pain management outcomes. To understand the molecular mechanisms of opioids, molecular signatures that arise from opioid exposure are often sought after, using various analytical methods. In this study, we performed proteomics, and multiglycomics via sequential analysis of polysialic acids, glycosaminoglycans, N-glycans and O-glycans, using the same cerebral spinal fluid (CSF) s le from patients that had long-term (& years), intrathecal morphine or baclofen administered via an indwelling pump. Proteomics and N-glycomics signatures between the two treatment groups were highly conserved, while significant differences were observed in polysialic acid, heparan sulfate glycosaminoglycan and O-glycan profiles between the two treatment groups. This represents the first study to investigate the potential relationships between erse CSF conjugated glycans and long-term intrathecal drug exposure. The unique changes, observed by a sequential analytical workflow, reflect previously undescribed molecular effects of opioid administration and pain management.

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

Publisher: Springer Science and Business Media LLC

Date: 04-2011

DOI: 10.1186/GB-2011-12-4-R40

Abstract: Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea / Trichoderma . Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis ) and Trichoderma virens (formerly Gliocladium virens , teleomorph Hypocrea virens ), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina ). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride . The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei . The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Transition of Mesenchymal and Epithelial Cancer Cells Depends on α1-4 Galactosyltransferase-Mediated Glycosphingolipids

Publisher: American Association for Cancer Research (AACR)

Date: 31-05-2018

DOI: 10.1158/0008-5472.CAN-17-2223

Abstract: The reversible transitions of cancer cells between epithelial and mesenchymal states comprise cellular and molecular processes essential for local tumor growth and respective dissemination. We report here that globoside glycosphingolipid (GSL) glycosyltransferase-encoding genes are elevated in epithelial cells and correlate with characteristic EMT signatures predictive of disease outcome. Depletion of globosides through CRISPR-Cas9–mediated deletion of the key enzyme A4GALT induces EMT, enhances chemoresistance, and increased CD24low/CD44high cells. The cholera toxin–induced mesenchymal-to-epithelial transition occurred only in cells with functional A4GALT. Cells undergoing EMT lost E-cadherin expression through epigenetic silencing at the promoter region of CDH1. However, in ΔA4GALT cells, demethylation was able to rescue E-cadherin–mediated cell–cell adhesion only in the presence of exogenous A4GALT. Overall, our data suggest another class of biomolecules vital for epithelial cancer cells and for maintaining cell integrity and function. Significance: This study highlights the essential role of glycosphingolipids in the maintenance of epithelial cancer cell properties. Cancer Res 78(11) 2952–65. ©2018 AACR.

Light‐independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Publisher: Wiley

Date: 07-1985

DOI: 10.1111/J.1399-3054.1985.TB03351.X

Effect of Tween 80 on the morphology of Trigonopsis variabilis

Publisher: Microbiology Society

Date: 08-1977

DOI: 10.1099/00221287-101-2-233

MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed paraffin-embedded murine kidney

Publisher: Springer Science and Business Media LLC

Date: 02-12-2015

DOI: 10.1007/S00216-014-8293-7

Differential involvement of glycans in the binding of Staphylococcus epidermidis and Corynebacterium spp. to human sweat

Publisher: Elsevier BV

Date: 04-1970

DOI: 10.1016/J.MICRES.2018.12.007

Abstract: Sweat is a secretory fluid that can be a source of unpleasant body odour due to interaction of resident bacteria with sweat components. Identification of glycoproteins in sweat suggests that protein-conjugated glycans may act as binding epitopes for bacteria, as found in other secretory fluids such as human milk, tears and saliva which help to protect epithelial surfaces from infection. We conducted proteomic and glycomic analysis of sweat to reveal an abundance of glycoproteins, predominantly carrying bi-antennary sialylated N-glycans with or without fucose. A fluorescent plate assay was used to determine whether glycans on sweat proteins provide binding epitopes for odour-producing skin commensals Staphylococcus epidermidis and Corynebacterium. Sialic acid and fucose were found to be important binding epitopes for S. epidermidis 3-22-BD-6, a strain recently isolated from human sweat, whereas fucose (but not sialic acid) contributed to the binding of Type strain S. epidermidis ATCC 12228. In contrast, our results indicate that sweat N-glycans do not provide binding epitopes for Corynebacterium. Synthetic sugar mimics of Lewis blood group antigens were investigated as potential inhibitors of the binding of S. epidermidis 3-22-BD-6 to sweat. Pre-incubation of the bacterium with LeB, LeX, LeY and sLeX (pentaose) resulted in a significant reduction in sweat protein adhesion indicating that terminal fucose is a key binding epitope, particularly when linked to a Type 2 chain (Galβ1-4GlcNAc) configuration (LeY). Our results form an impetus for future studies seeking to elucidate the role of glycans in sweat associated malodour, with possible implications for cosmetic and medical fields.

Rapid and sensitive glycan targeting by lectin-SERS assay

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/C9MO00181F

Abstract: The fabrication of lectin-SERS nanotags and the assay designed for rapid glycoprotein identification and quantification.

Glycomic Mass Spectrometric Analysis and Data Interpretation Tools

Publisher: Wiley

Date: 13-11-2009

DOI: 10.1002/9780470029619.CH12

New urinary EPO drug testing method using two-dimensional gel electrophoresis

Publisher: Elsevier BV

Date: 08-2005

DOI: 10.1016/J.CCCN.2005.03.013

Abstract: We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection. The 2DE method separates HuEPO and rHuEPO isoforms by both iso-electric point and molecular mass. We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO however, they separate distinctly by the 2DE method. Alpha-2-HS-glycoprotein (HSGP) was identified by peptide mass fingerprinting as one of the urinary cross-reacting proteins, and commercially available purified HSGP was chosen to be added into urine s les as an internal standard prior to separation. Software (EpIQ) was specifically developed that applies four separate criteria to the detection of the migration of rHuEPO and HuEPO relative to the internal standard. The combination of s le preparation, two-dimensional separation, internal standard, standardized blotting procedures and image analysis software enables the 2DE test for rHuEPO in urine to be performed reproducibly and accurately.

Cancer-derived small extracellular vesicles: emerging biomarkers and therapies for pancreatic ductal adenocarcinoma diagnosis/prognosis and treatment

Publisher: Springer Science and Business Media LLC

Date: 14-10-2022

DOI: 10.1186/S12951-022-01641-0

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers worldwide with high mortality, which is mainly due to the lack of reliable biomarkers for PDAC diagnosis rognosis in the early stages and effective therapeutic strategies for the treatment. Cancer-derived small extracellular vesicles (sEVs), which carry various messages and signal biomolecules (e.g. RNAs, DNAs, proteins, lipids, and glycans) to constitute the key features (e.g. genetic and phenotypic status) of cancer cells, are regarded as highly competitive non-invasive biomarkers for PDAC diagnosis rognosis. Additionally, new insights on the biogenesis and molecular functions of cancer-derived sEVs pave the way for novel therapeutic strategies based on cancer-derived sEVs for PDAC treatment such as inhibition of the formation or secretion of cancer-derived sEVs, using cancer-derived sEVs as drug carriers and for immunotherapy. This review provides a comprehensive overview of the most recent scientific and clinical research on the discovery and involvement of key molecules in cancer-derived sEVs for PDAC diagnosis rognosis and strategies using cancer-derived sEVs for PDAC treatment. The current limitations and emerging trends toward clinical application of cancer-derived sEVs in PDAC diagnosis rognosis and treatment have also been discussed.

Updates to the Symbol Nomenclature for Glycans guidelines

Publisher: Oxford University Press (OUP)

Date: 11-06-2019

DOI: 10.1093/GLYCOB/CWZ045

Abstract: The Symbol Nomenclature for Glycans (SNFG) is a community-curated standard for the depiction of monosaccharides and complex glycans using various colored-coded, geometric shapes, along with defined text additions. It is hosted by the National Center for Biotechnology Information (NCBI) at the NCBI-Glycans Page (lycans/snfg.html). Several changes have been made to the SNFG page in the past year to update the rules for depicting glycans using the SNFG, to include more ex les of use, particularly for non-mammalian organisms, and to provide guidelines for the depiction of ambiguous glycan structures. This Glycoforum article summarizes these recent changes.

Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum: In vivo studies on glycosylation of mucin MUC1 and MUC2 repeats

Publisher: Wiley

Date: 15-04-1998

DOI: 10.1046/J.1432-1327.1998.2530517.X

Abstract: One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Ser and Thr residues on secreted or membrane-bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc :polypeptide N-acetylglucosaminyltransferase(s). Glutathione S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2-like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in alpha configuration as determined by NMR studies.

Comparison of Methods for Profiling O-Glycosylation

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1074/MCP.M900450-MCP200

Navigating the Glycome Space and Connecting the Glycoproteome

Publisher: Springer New York

Date: 2017

DOI: 10.1007/978-1-4939-6783-4_7

Abstract: UniCarbKB ( unicarbkb.org ) is a comprehensive resource for mammalian glycoprotein and annotation data. In particular, the database provides information on the oligosaccharides characterized from a glycoprotein at either the global or site-specific level. This evidence is accumulated from a peer-reviewed and manually curated collection of information on oligosaccharides derived from membrane and secreted glycoproteins purified from biological fluids and/or tissues. This information is further supplemented with experimental method descriptions that summarize important s le preparation and analytical strategies. A new release of UniCarbKB is published every three months, each includes a collection of curated data and improvements to database functionality. In this Chapter, we outline the objectives of UniCarbKB, and describe a selection of step-by-step workflows for navigating the information available. We also provide a short description of web services available and future plans for improving data access. The information presented in this Chapter supplements content available in our knowledgebase including regular updates on interface improvements, new features, and revisions to the database content ( confluence.unicarbkb.org ).

Protein phosphorylation

Publisher: Elsevier BV

Date: 05-1998

DOI: 10.1016/S0021-9673(98)00115-0

Abstract: Protein phosphorylation plays a central role in many biological and biomedical phenomena. In this review, while a brief overview of the occurrence and function of protein phosphorylation is given, the primary focus is on studies related to the detection and analysis of phosphorylation both in vivo and in vitro. We focus on phosphorylation of serine, threonine and tyrosine, the most commonly phosphorylated amino acids in eukaryotes. Technologies such as radiolabelling, antibody recognition, chromatographic methods (HPLC, TLC), electrophoresis, Edman sequencing and mass spectrometry are reviewed. We consider the speed, simplicity and sensitivity of tools for detection and identification of protein phosphorylation, as well as quantitation and site characterisation. The limitations of currently available methods are summarised.

Small-Scale Analysis of O-Linked Oligosaccharides from Glycoproteins and Mucins Separated by Gel Electrophoresis

Publisher: American Chemical Society (ACS)

Date: 17-10-2002

DOI: 10.1021/AC025890A

Abstract: A technique with subpicomolar sensitivity was developed for analyzing O-linked oligosaccharides released from glycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly-(vinylidene fluoride) membrane, reductive beta-elimination, and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray mass spectrometry. It was also found that N-linked oligosaccharides could be recovered under the same conditions, found both as free oligosaccharides and as distinct glycopeptides created from reductive cleavage of the protein backbone, giving some information on site-specific glycosylation. The method was used to demonstrate that the difference between human alpha-2HS-glycoprotein isoforms separated by 2D-gel electrophoresis was partially due to sialylation of both O-linked and N-linked oligosaccharides. It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecular mass (200,000-5,000,000 Da) highly glycosylated mucin glycoproteins collected from small intestine and saliva and separated by sodium dodecyl sulfate-agarose olyacrylamide composite gels. Mass spectrometric data not only gave information about the mass distribution of the heterogeneous mixtures of oligosaccharides from [M - xH](x-) ions but also gave information about the isomeric heterogeneity of the oligosaccharides from their resolution by porous graphitized carbon chromatography. Tandem mass spectrometry was explored as a technique for distinguishing between oligosaccharide isomers with different sequences and also between oligosaccharides with the same sequence but with different linkage configurations.

Frontiers in glycomics: Bioinformatics and biomarkers in disease An NIH White Paper prepared from discussions by the focus groups at a workshop on the NIH campus, Bethesda MD (September 11–13, 2006)

Publisher: Wiley

Date: 2008

DOI: 10.1002/PMIC.200700917

Abstract: Key issues relating to glycomics research were discussed after the workshop entitled "Frontiers in Glycomics: Bioinformatics and Biomarkers in Disease" by two focus groups nominated by the organizers. The groups focused on two themes: (i) glycomics as the new frontier for the discovery of biomarkers of disease and (ii) requirements for the development of informatics for glycomics and glycobiology. The mandate of the focus groups was to build consensus on these issues and develop a summary of findings and recommendations for presentation to the NIH and the greater scientific community. A list of scientific priorities was developed, presented, and discussed at the workshops. Additional suggestions were solicited from workshop participants and collected using the workshop mailing list. The results are summarized in this White Paper, authored by the co-chairs of the focus groups.

Development of a mass fingerprinting tool for automated interpretation of oligosaccharide fragmentation data

Publisher: Wiley

Date: 25-05-2004

DOI: 10.1002/PMIC.200300784

Abstract: The bioinformatic tool GlycosidIQ was developed for computerized interpretation of oligosaccharide mass spectrometric fragmentation based on matching experimental data with theoretically fragmented oligosaccharides generated from the database GlycoSuiteDB. This use of the software for glycofragment mass fingerprinting obviates a large part of the manual, labor intensive, and technically challenging interpretation of oligosaccharide fragmentation. Using 130 negative ion electrospray ionization-tandem mass spectrometry fragment spectra from identified oligosaccharide structures, it was shown that the GlycosidIQ scoring algorithms were able to correctly identify oligosaccharides in the great majority of cases (correct structure top ranked in 78% of the cases and an additional 17% were ranked second highest in the s le set).

Glyco-scope into the role of protein glycosylation in the female reproductive tract

Publisher: Forum: Carbohydrates Coming of Age

Date: 25-03-2020

DOI: 10.4052/TIGG.1820.1E

Tissue glycomics distinguish tumour sites in women with advanced serous adenocarcinoma.

Publisher: Wiley

Date: 29-09-2017

DOI: 10.1002/1878-0261.12134

Databases and Associated Tools for Glycomics and Glycoproteomics

Publisher: Springer New York

Date: 15-10-2016

DOI: 10.1007/978-1-4939-6493-2_18

Abstract: The access to biodatabases for glycomics and glycoproteomics has proven to be essential for current glycobiological research. This chapter presents available databases that are devoted to different aspects of glycobioinformatics. This includes oligosaccharide sequence databases, experimental databases, 3D structure databases (of both glycans and glycorelated proteins) and association of glycans with tissue, disease, and proteins. Specific search protocols are also provided using tools associated with experimental databases for converting primary glycoanalytical data to glycan structural information. In particular, researchers using glycoanalysis methods by U/HPLC (GlycoBase), MS (GlycoWorkbench, UniCarb-DB, GlycoDigest), and NMR (CASPER) will benefit from this chapter. In addition we also include information on how to utilize glycan structural information to query databases that associate glycans with proteins (UniCarbKB) and with interactions with pathogens (SugarBind).

Small-Scale Enzymatic Digestion of Glycoproteins and Proteoglycans for Analysis of Oligosaccharides by LC-MS and FACE Gel Electrophoresis

Publisher: Humana Press

Date: 20-02-2008

DOI: 10.1007/978-1-59745-022-5_13

Abstract: Structural characterization of oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel electrophoresis. S le preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked oligosaccharides. In addition, accompanying procedures for oligosaccharide reduction by sodium borohydride, s le desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale oligosaccharide species fractionation are included. Fluorophore-assisted carbohydrate electrophoresis (FACE) is another useful method to isolate derivatized oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.

The Hitchhiker's guide to glycoproteomics

Publisher: Portland Press Ltd.

Date: 20-07-2021

DOI: 10.1042/BST20200879

Abstract: Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. This ‘Hitchhiker's guide to glycoproteomics’ is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. Ex les of the variety of approaches, methodologies, and technologies currently used in the field are given. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins — a class of biomolecules essential in regulating cell function.

The effect of streptozotocin-induced hyperglycemia on N-and O-linked protein glycosylation in mouse ovary

Publisher: Oxford University Press (OUP)

Date: 28-08-2018

DOI: 10.1093/GLYCOB/CWY075

Abstract: Post-translational modification of proteins namely glycosylation influences cellular behavior, structural properties and interactions including during ovarian follicle development and atresia. However, little is known about protein glycosylation changes occurring in diabetes mellitus in ovarian tissues despite the well-known influence of diabetes on the outcome of successful embryo implantation. In our study, the use of PGC chromatography-ESI mass spectrometry in negative ion mode enabled the identification of 138 N-glycans and 6 O-glycans on the proteins of Streptozotocin-induced (STZ) diabetic mouse ovarian tissues (n = 3). Diabetic mouse ovaries exhibited a relative decrease in sialylation, fucosylation and, to a lesser extent, branched N-linked glycan structures, as well as an increase in oligomannose structures on their proteins, compared with nondiabetic mouse ovaries. Changes in N-glycans occurred in the diabetic liver tissue but were more evident in diabetic ovarian tissue of the same mouse, suggesting an organ-specific effect of diabetes mellitus on protein glycosylation. Although at a very low amount, O-GalNAc glycans of mice ovaries were present as core type 1 and core type 2 glycans with a relative increase in the NeuGc:NeuAc ratio as the most significant difference between control and diabetic ovarian tissues. STZ-treated mice also showed a trend towards an increase in TNF-α and IL1-B inflammatory cytokines, which have previously been shown to influence protein glycosylation.

GlycoSpectrumScan

Publisher: American Chemical Society (ACS)

Date: 19-01-2010

DOI: 10.1021/PR900956X

Abstract: With the emergence of glycoproteomics, there is a need to develop bioinformatic tools to identify glycopeptides in protease digests of glycoproteins. GlycoSpectrumScan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. Two experimental data sets are required as inputs: (1) oligosaccharide compositions of the N- and/or O-linked glycans present in the s le and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of N- and/or O-glycosylation sites. GlycoSpectrumScan uses MS data, rather than MS/MS data, to identify glycopeptides and determine the relative distribution of N- and O-glycoforms at each site. It is functional for assigning monosaccharide compositions on glycopeptides with single and multiple sites of glycosylation. The algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both ESI and MALDI data from all mass spectrometer platforms. Manual analysis time for identifying glycosylation heterogeneity at each site on glycoprotein(s) is substantially decreased. The application of this tool to characterize the N- and O-linked glycopeptides from human secretory IgA (sIgA), consisting of secretory component (7 N-linked sites), IgA1 (2 N-linked, <or=5 O-linked sites), IgA2 (4 N-linked sites) and J-chain (1 N-linked site) is described. GlycoSpectrumScan is freely available at www.glycospectrumscan.org .

Modification of Asparagine-Linked Glycan Density for the Design of Hepatitis B Virus Virus-Like Particles with Enhanced Immunogenicity

Publisher: American Society for Microbiology

Date: 15-11-2015

DOI: 10.1128/JVI.01123-15

Abstract: The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified virus-like particles (VLPs) exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wild-type (WT) HBsAgS N146 glycosylation site was converted to N146Q for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced T116N and G130N sites were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs, with minor variations regarding the degree of fucosylation, bisecting N -acetylglucosamines, and sialylation. Antigenic fingerprints for the WT and hypo- and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their ability to bind to the different VLP glyco-analogues, suggesting that the additional N -glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N -glycan abundance and antibody titers. The T116N VLPs induced earlier and longer-lasting antibody responses than did the hypoglycosylated and WT VLPs. The ability of nonnative VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. IMPORTANCE The use of biochemically modified, nonnative immunogens represents an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N -glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized, and the results demonstrated that they carry the same types of glycans as wild-type VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N -glycan density induce earlier and longer-lasting antibody immune responses than do wild-type or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.

Engineering 3D living brain tumour equivalents as macroscopic test-systems for the development of optical imaging and sensing applications (Conference Presentation)

Publisher: SPIE

Date: 04-03-2019

DOI: 10.1117/12.2509582

e-workflow for recording of glycomic mass spectrometric data in compliance with reporting guidelines

Publisher: Cold Spring Harbor Laboratory

Date: 27-08-2018

DOI: 10.1101/401141

Abstract: Glycomics targets released glycans from proteins, lipids and proteoglycans. High throughput glycomics is based on mass spectrometry (MS) that increasingly depends on exchange of data with databases and the use of software. This requires an agreed format for accurately recording of experiments, developing consistent storage modules and granting public access to glycomic MS data. The introduction of the MIRAGE (Mimimum Requirement for A Glycomics Experiment) reporting standards for glycomics was the first step towards automating glycomic data recording. This report describes a glycomic e-infrastructure utilizing a well established glycomics recording format (GlycoWorkbench), and a dedicated web tool for submitting MIRAGE-compatible MS information into a public experimental repository, UniCarb-DR. The submission of data to UniCarb-DR should be a part of the submission process for publications with glycomics MS n that conform to the MIRAGE guidelines. The structure of this pipeline allows submission of most MS workflows used in glycomics.

BOLD - A biological O-linked glycan database

Publisher: Wiley

Date: 12-1999

DOI: 10.1002/(SICI)1522-2683(19991201)20:18<3589::AID-ELPS3589>3.0.CO;2-M

Autoimmue hepatitis (AIH) and primary biliary cirrhosis (PBC) alter the N-linked glycosylation pathway

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1016/S0168-8278(15)31384-2

ESGO State of the Art Conference, Turin, Italy: Follow up in Gynaecological Malignancies

Publisher: BMJ

Date: 09-2014

DOI: 10.1097/01.IGC.0000455561.57987.EA

Phenotyping CHST3 skeletal dysplasia from freezer-induced urine sediments

Publisher: CSIRO Publishing

Date: 31-05-2023

DOI: 10.1071/CH23041

Abstract: Skeletal dysplasias are a group of rare genetic disorders that affect growth and development of the skeleton, leading to physical deformities and other medical problems. High-throughput genome sequencing technologies have made it easier to genotype the disorder, but do not always reflect the phenotypic outcome. CHST3-related skeletal dysplasia is caused by the reduced function of the carbohydrate sulfotransferase that sulfates chondroitin sulfate glycosaminoglycans. We show in this pilot study that we were able to phenotype patients with CHST3-related skeletal dysplasia by profiling the glycosaminoglycans and identifying their potential protein carriers sequentially using freezer-induced patient urine sediments.

Cereal products derived from wheat, sorghum, rice and oats alter the infant gut microbiota in vitro

Publisher: Springer Science and Business Media LLC

Date: 30-10-2017

DOI: 10.1038/S41598-017-14707-Z

Abstract: The introduction of different nutrient and energy sources during weaning leads to significant changes in the infant gut microbiota. We used an in vitro infant digestive and gut microbiota model system to investigate the effect of four commercially available cereal products based on either wheat, sorghum, rice or oats, on the gut microbiota of six infants. Our results indicated cereal additions induced numerous changes in the gut microbiota composition. The relative abundance of bacterial families associated with fibre degradation, Bacteroidaceae , Bifidobacteriaceae , Lactobacillaceae , Prevotellaceae , Ruminococcaceae and Veillonellaceae increased, whilst the abundance of Enterobacteriaceae decreased with cereal additions. Corresponding changes in the production of SCFAs showed higher concentrations of acetate following all cereal additions, whilst, propionate and butyrate varied between specific cereal additions. These cereal-specific variations in the concentrations of SCFAs showed a moderate correlation with the relative abundance of potential SCFA-producing bacterial families. Overall, our results demonstrated clear shifts in the abundance of bacterial groups associated with weaning and an increase in the production of SCFAs following cereal additions.

Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants

Publisher: Oxford University Press (OUP)

Date: 07-11-2011

DOI: 10.1093/GLYCOB/CWR157

A Sydney proteome story

Publisher: Elsevier BV

Date: 07-2014

DOI: 10.1016/J.JPROT.2014.04.006

Abstract: This is the story of the experience of a multidisciplinary group at Macquarie University in Sydney as we participated in, and impacted upon, major currents that washed through protein science as the field of Proteomics emerged. The large scale analysis of proteins became possible. This is not a history of the field. Instead we have tried to encapsulate the stimulating personal ride we had transiting from conventional academe, to a Major National Research Facility, to the formation of Proteomics company Proteome Systems Ltd. There were lots of blind alleys, wrong directions, but we also got some things right and our efforts, along with those of many other groups around the world, did change the face of protein science. While the transformation is by no means yet complete, protein science is very different from the field in the 1990s. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears

Publisher: Portland Press Ltd.

Date: 09-2002

DOI: 10.1042/BJ20011876

Abstract: Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose olyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of & kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

Enabling Sensitive Phenotypic Profiling of Cancer-Derived Small Extracellular Vesicles Using Surface-Enhanced Raman Spectroscopy Nanotags

Publisher: American Chemical Society (ACS)

Date: 05-03-2020

DOI: 10.1021/ACSSENSORS.9B02377

Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine κ-casein

Publisher: Oxford University Press (OUP)

Date: 1994

DOI: 10.1093/GLYCOB/4.6.837

Abstract: kappa-Casein is the major glycoprotein in bovine milk. It has a proteinase-sensitive (chymosin) site which cleaves the glycoprotein into two segments: N-terminal para-kappa-casein domain and the C-terminal kappa-casein macroglycopeptide domain which is highly heterogeneous in oligosaccharide content. We have identified six sites of O-glycosylation on the macroglycopeptide by solid-phase Edman degradation: Thr121, Thr131, Thr133, Thr136 (A variant only), Thr142 and Thr165. No Ser residues are glycosylated. The glycosylation status of 15 of 17 potential O-glycosylation sites in the B variant was accurately predicted using the four peptide motifis previously proposed for the glycosylation of human glycophorin A (Pisano, A., Redmond, J.W., Williams, K.L. and Gooley, A.A., Glycobiology, 3, 429-435, 1993), provided one additional assumption is made concerning an inhibitory role for a nearby Ile.

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

Publisher: Springer Science and Business Media LLC

Date: 1990

DOI: 10.1007/BF00030067

P1 glycosphingolipid is expressed on ovarian cancer cells recognized by naturally occurring anti-P1 antibodies

Publisher: American Association for Cancer Research (AACR)

Date: 10-2013

DOI: 10.1158/1078-0432.OVCA13-B23

Abstract: Using printed glycan array we have recently demonstrated that natural occurring anti-glycan antibodies to chemically synthesized P1 trisaccharide and Pk are significantly reduced in ovarian cancer patients (1). These findings were successfully validated with two in-house developed independent glycan-based immunoassays (ELISA and suspension array) (2). Both candidates are glycosphingolipids (GSL) and share similar structural motifs. P1 naturally occurs as a pentasaccharide belonging to the neolacto series. Here we aim to a) validate our findings in an independent validation cohort, b) identify the immunoglobulin subtype causing this observation, c) identify the corresponding naturally expressed P1 antigen in cancer cells, and c) purify human anti-P1 antibodies from cancer ascites. A cohort of 155 blood plasma s les comprising healthy controls and patients with cancer of the ovary, tube and peritoneum was profiled for P1 antibodies using monoplex suspension array. GSL isolated from fresh cancer tissue were PVDF blotted and extracted enzymatically following purification of glycan alditols. Glycan structures were identified by negative-mode LC-ESI-MS. P1-expressing cancer cell lines were detected using flow cytometry. Human anti-P1 antibodies were affinity purified from cancer ascites and studied using glycan-based immunoassays. Glycan-based immunoassay profiling of validation cohort confirmed our recent findings, reduced anti-P1 antibody levels in cancer patients compared to healthy controls (p=0.0002). We could also demonstrate that this observation was caused by IgM subtypes and not IgG. Corresponding P blood group- related antigens (GSL) including P1 and Pk were identified in cancer tissue s les. Flow cytometry revealed that a serous ovarian cancer cell line is positive for P1 (up to 46%). We further demonstrate that affinity purified anti-P1 antibodies bind naturally expressed P1 GSL on ovarian cancer cell surface. Monoclonal as well as affinity purified anti-P1 antibodies applied to identify P1-expressing cancer cell lines revealed high specificity to P1 shown by inhibition assay and glycan-based immunoassays. In this study we demonstrated anti-P1 IgM levels are decreased in a significant number of cancer patients and that the GSL P1 is present on ovarian/ peritoneal cancer cells, both in tissue and immortalized cell lines. We can therefore conclude that P1 is an epitope for human circulating anti-glycan antibodies. These results are in concordance with our previous findings and support the presence of a new antigenic tumor-associated GSL in ovarian cancer. The study of the molecular profile leading to the presence of P1 in ovarian cancer cells is presently under investigation. 1. Jacob F, et al. (2012) Serum antiglycan antibody detection of nonmucinous ovarian cancers by using a printed glycan array. International Journal of Cancer 130(1):138-146. 2. Pochechueva T, et al. (2011) Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies. Glycoconjugate journal 28(8-9):507-517. Citation Format: Francis Jacob, Merrina Anugraham, Alam Shahidul, Nicolai Bovin, Nicole Packer, Neville Hacker, Viola Heinzelmann-Schwarz. P1 glycosphingolipid is expressed on ovarian cancer cells recognized by naturally occurring anti-P1 antibodies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic Sep 18-21, 2013 Miami, FL. Philadelphia (PA): AACR Clin Cancer Res 2013 (19 Suppl):Abstract nr B23.

Relative quantitation of multi-antennary N-glycan classes: combining PGC-LC-ESI-MS with exoglycosidase digestion

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C5AN00691K

Abstract: In the search for N -glycan disease biomarkers current glycoanalytical methods may not be revealing a complete picture of precious s les, and we may be missing valuable structural information that fall outside analysis windows.

Ovarian cancer cells specifically express LacdiNAc and show elevated B4GALNT3/4 expression

Publisher: Oxford University Press (OUP)

Date: 06-10-2015

DOI: 10.1093/GLYCOB/CWV068

Chemoenzymatic glycan labelling as a platform for site-specific IgM-antibody drug conjugates

Publisher: Elsevier BV

Date: 11-2019

DOI: 10.1016/J.AB.2019.113385

Abstract: Immunoglobulin M (IgM) type antibodies play a significant role in complement activation, cellular debris clearance and cell quality control, and have the potential to be used as a therapeutic or targeting/delivery antibody. However, this potential has not been explored thoroughly due to its high molecular weight, polymeric structure and large number of glycosylation sites. Site-specific antibody-drug-conjugates (ADC) are considered the next generation protein biotherapeutic drugs and currently all, in clinical trials and approved, are of the IgG isotype. As existing methods for the development and characterization of IgG-ADCs are not compatible with IgM-ADC, we describe a platform methodology suitable for site specific IgM-ADC using a chemoenzymatic method targeting the glycans on the IgM. Azide functionalized sialic acids were incorporated onto IgM glycans using sialyltransferase for biocompatible conjugation using "click" chemistry. The number of azide groups incorporated onto the IgM glycans were characterized by mass spectrometry of the enzymatically released glycans and glycopeptides. Quantitation of the azide incorporation showed an azide antibody ratio of 8 (glycan data) and 6-10 (glycopeptide data) which translates to a high drug antibody ratio based on IgG-ADC standards. This platform methodology can be readily adapted for any human IgM produced in a mammalian cell expression system.

Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization

Publisher: Springer Science and Business Media LLC

Date: 21-03-2019

DOI: 10.1186/S12974-019-1451-2

Use of graphitised carbon negative ion LC–MS to analyse enzymatically digested glycosaminoglycans

Publisher: Elsevier BV

Date: 25-09-2005

DOI: 10.1016/J.JCHROMB.2005.07.014

Abstract: Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and s le preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid s les.

Building a PGC-LC-MS N-glycan retention library and elution mapping resource

Publisher: Springer Science and Business Media LLC

Date: 13-09-2017

DOI: 10.1007/S10719-017-9793-4

Abstract: Porous graphitised carbon-liquid chromatography (PGC-LC) has been proven to be a powerful technique for the analysis and characterisation of complex mixtures of isomeric and isobaric glycan structures. Here we evaluate the elution behaviour of N-glycans on PGC-LC and thereby provide the potential of using chromatographic separation properties, together with mass spectrometry (MS) fragmentation, to determine glycan structure assignments more easily. We used previously reported N-glycan structures released from the purified glycoproteins Immunoglobulin G (IgG), Immunoglobulin A (IgA), lactoferrin, α1-acid glycoprotein, Ribonuclease B (RNase B), fetuin and ovalbumin to profile their behaviour on capillary PGC-LC-MS. Over 100 glycan structures were determined by MS/MS, and together with targeted exoglycosidase digestions, created a N-glycan PGC retention library covering a full spectrum of biologically significant N-glycans from pauci mannose to sialylated tetra-antennary classes. The resultant PGC retention library ( howPgc ) incorporates retention times and supporting fragmentation spectra including exoglycosidase digestion products, and provides detailed knowledge on the elution properties of N-glycans by PGC-LC. Consequently, this platform should serve as a valuable resource for facilitating the detailed analysis of the glycosylation of both purified recombinant, and complex mixtures of, glycoproteins using established workflows.

UniCarbKB: Emergent Knowledgebase for Glycomics

Publisher: Springer Japan

Date: 20-10-2014

DOI: 10.1007/978-4-431-54841-6_21

Enzymatic Azido-GalNAc-Functionalized Silk Fibroin for Click Chemistry Conjugation

Publisher: American Chemical Society (ACS)

Date: 25-03-2021

DOI: 10.1021/ACS.BIOMAC.0C01791

Production of α-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease

Publisher: Springer Science and Business Media LLC

Date: 18-09-2012

DOI: 10.1038/NCOMMS2070

Abstract: Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzyme replacement therapy. Towards developing a plant-based enzyme replacement therapeutic for the lysosomal storage disease mucopolysaccharidosis I, here we expressed α-L-iduronidase in the endosperm of maize seeds by a previously uncharacterized mRNA-targeting-based mechanism. Immunolocalization, cellular fractionation and in situ RT-PCR demonstrate that the α-L-iduronidase protein and mRNA are targeted to endoplasmic reticulum (ER)-derived protein bodies and to protein body-ER regions, respectively, using regulatory (5'- and 3'-UTR) and signal-peptide coding sequences from the γ-zein gene. The maize α-L-iduronidase exhibits high activity, contains high-mannose N-glycans and is amenable to in vitro phosphorylation. This mRNA-based strategy is of widespread importance as plant N-glycan maturation is controlled and the therapeutic protein is generated in a native form. For our target enzyme, the N-glycan structures are appropriate for downstream processing, a prerequisite for its potential as a therapeutic protein.

Lipids and the morphogenesis of Trigonopsis variabilis

Publisher: Oxford University Press (OUP)

Date: 10-1981

DOI: 10.1111/J.1574-6968.1981.TB07627.X

SugarBindDB: Resource of Pathogen Lectin-Glycan Interactions

Publisher: Springer Japan

Date: 20-10-2014

DOI: 10.1007/978-4-431-54841-6_28

Comparative structural analysis of the glycosylation of salivary and buccal cell proteins: innate protection against infection by Candida albicans

Publisher: Oxford University Press (OUP)

Date: 24-07-2012

DOI: 10.1093/GLYCOB/CWS112

Abstract: Mucosal epithelial surfaces, such as line the oral cavity, are common sites of microbial colonization by bacteria, yeast and fungi. The microbial interactions involve adherence between the glycans on the host cells and the carbohydrate-binding proteins of the pathogen. Saliva constantly bathes the buccal cells of the epithelial surface of the mouth and we postulate that the sugars on the salivary glycoproteins provide an innate host immune mechanism against infection by competitively inhibiting pathogen binding to the cell membranes. The structures of the N- and O-linked oligosaccharides on the glycoproteins of saliva and buccal cell membranes were analyzed using capillary carbon liquid chromatography-electrospray ionization MS/MS. The 190 glycan structures that were characterized were qualitatively similar, but differed quantitatively, between saliva and epithelial buccal cell membrane proteins. The similar relative abundance of the terminal glycan epitope structures (e.g. ABO(H) blood group, sialylation and Lewis-type antigens) on saliva and buccal cell membrane glycoproteins indicated that the terminal N- and O-linked glycan substructures in saliva could be acting as decoy-binding receptors to competitively inhibit the attachment of pathogens to the surface of the oral mucosa. A flow cytometry-based binding assay quantified the interaction between buccal cells and the commensal oral pathogen Candida albicans. Whole saliva and released glycans from salivary proteins inhibited the interaction of C. albicans with buccal epithelial cells, confirming the protective role of the glycans on salivary glycoproteins against pathogen infection.

Analysis of mucosal mucins separated by SDS‐urea agarose polyacrylamide composite gel electrophoresis

Publisher: Wiley

Date: 24-11-2011

DOI: 10.1002/ELPS.201100374

Abstract: Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose olyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin s les. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.

Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching

Publisher: Oxford University Press (OUP)

Date: 13-07-2012

DOI: 10.1093/GLYCOB/CWS110

Abstract: Growing evidence indicates that the in idualized and highly reproducible N-glycan repertoires on each protein glycosylation site modulate function. Relationships between protein structures and the resulting N-glycoforms have previously been observed, but remain to be quantitatively confirmed and examined in detail to define the responsible mechanisms in the conserved mammalian glycosylation machinery. Here, we investigate this relationship by manually extracting and analyzing quantitative and qualitative site-specific glycoprofiling data from 117 research papers. Specifically, N-glycan structural motifs were correlated with the structure of the protein carriers, focusing on the solvent accessibility of the in idual glycosylation sites and the physicochemical properties of the surrounding polypeptide chains. In total, 474 glycosylation sites from 169 mammalian N-glycoproteins originating from different tissues/body fluids were investigated. It was confirmed statistically that the N-glycan type, degree of core fucosylation and branching are strongly influenced by the glycosylation site accessibility. For these three N-glycan features, glycosylation sites carrying highly processed glycans were significantly more solvent-accessible than those carrying less processed counterparts. The glycosylation site accessibilities could be linked to molecular signatures at the primary and secondary protein levels, most notably to the glycoprotein size and the proportion of glycosylation sites located in accessible β-turns. In addition, the subcellular location of the glycoproteins influenced the formation of the N-glycan structures. These data confirm that protein structures dictate site-specific formation of several features of N-glycan structures by affecting the biosynthetic pathway. Mammals have, as such, evolved mechanisms enabling proteins to influence the N-glycans they present to the extracellular environment.

Identification and characterization of glycosylated phenylthiohydantoin amino acids

Publisher: Springer US

Date: 1995

DOI: 10.1007/978-1-4899-1031-8_7

Cystic fibrosis and bacterial colonization define the sputum N-glycosylation phenotype

Publisher: Oxford University Press (OUP)

Date: 04-09-2014

DOI: 10.1093/GLYCOB/CWU092

Abstract: Although mucin O-glycosylation of sputum from in iduals suffering from cystic fibrosis (CF) is known to be altered relative to their unaffected counterparts, protein N-glycosylation of CF sputum remains structurally and functionally under-characterized. We report the first N-glycome of soluble proteins in sputum derived from five CF patients, two pathogen-free and two pathogen-infected/colonized non-CF in iduals suffering from other pulmonary conditions. N-Glycans were profiled using porous graphitized carbon-liquid chromatography-negative ion-tandem mass spectrometry following enzymatic release from sputum proteins. The composition, topology and linkage isomers of 68 N-glycans were characterized and relatively quantified. Recurring structural features in all sputum N-glycomes were terminal α2,6-sialylation, α1,6-core fucosylation, β1,4-bisecting GlcNAcylation and compositions indicating paucimannosylation. Despite covering different genotypes, age, gender and microbial flora, the sputum N-glycomes showed little interpatient and longitudinal variation within CF patients. Comparative N-glycome analysis between inter-patient group revealed that lung infection/colonization, in general, extensively enriches the CF sputum N-glycosylation phenotype with paucimannose with simultaneous over-sialylation/fucosylation and under-bisecting GlcNAcylation of complex/hybrid N-glycans. In contrast, the sputum from CF patients had only slightly increased abundance of paucimannose N-glycans relative to pathogen-infected/colonized non-CF in iduals. Similar to mucin O-glycosylation, protein N-glycosylation appears to be regulated primarily as a secondary effect of bacterial infection and inflammation rather than the CF pathogenesis in sputum. This study provides new structural N-glycan information to help understand the complex cellular and molecular environment of the CF affected respiratory tract.

Bisecting GlcNAc protein N-glycosylation is characteristic of human adipogenesis

Publisher: American Chemical Society (ACS)

Date: 31-12-2021

DOI: 10.1021/ACS.JPROTEOME.0C00702

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Publisher: Wiley

Date: 29-10-2012

DOI: 10.1002/PMIC.201200354

Abstract: Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid-sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site-specific characterization of the N- and O-linked glycosylation of serum-derived hSHBG. MS-driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom-up and top-down manner to determine glycosylation sites, site-specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum-derived hSHBG is N-glycosylated at Asn(351) and Asn(367) with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi- and triantennary N-Glycoforms with lactosamine-type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N-Glycoforms of Asn(367) were slightly more branched and core fucosylated than Asn(351) N-glycoforms due probably to a more surface-exposed glycosylation site. The N-terminal Thr(7) was fully occupied by the two O-linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn(351) glycan occupancies and N-glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.

The elimination of O-linked glycans from glycoproteins under non-reducing conditions

Publisher: Springer Science and Business Media LLC

Date: 04-1994

DOI: 10.1007/BF00731156

Development of a data independent acquisition mass spectrometry workflow to enable glycopeptide analysis without predefined glycan compositional knowledge

Publisher: Elsevier BV

Date: 02-2018

DOI: 10.1016/J.JPROT.2017.10.011

Abstract: Glycoproteomics investigates glycan moieties in a site specific manner to reveal the functional roles of protein glycosylation. Identification of glycopeptides from data-dependent acquisition (DDA) relies on high quality MS/MS spectra of glycopeptide precursors and often requires manual validation to ensure confident assignments. In this study, we investigated pseudo-MRM (MRM-HR) and data-independent acquisition (DIA) as alternative acquisition strategies for glycopeptide analysis. These approaches allow data acquisition over the full MS/MS scan range allowing data re-analysis post-acquisition, without data re-acquisition. The advantage of MRM-HR over DDA for N-glycopeptide detection was demonstrated from targeted analysis of bovine fetuin where all three N-glycosylation sites were detected, which was not the case with DDA. To overcome the duty cycle limitation of MRM-HR acquisition needed for analysis of complex s les such as plasma we trialed DIA. This allowed development of a targeted DIA method to identify N-glycopeptides without pre-defined knowledge of the glycan composition, thus providing the potential to identify N-glycopeptides with unexpected structures. This workflow was demonstrated by detection of 59 N-glycosylation sites from 41 glycoproteins from a HILIC enriched human plasma tryptic digest. 21 glycoforms of IgG1 glycopeptides were identified including two truncated structures that are rarely reported. We developed a data-independent mass spectrometry workflow to identify specific glycopeptides from complex biological mixtures. The novelty is that this approach does not require glycan composition to be pre-defined, thereby allowing glycopeptides carrying unexpected glycans to be identified. This is demonstrated through the analysis of immunoglobulins in human plasma where we detected two IgG1 glycoforms that are rarely observed.

Proteome analysis of glycoforms

Publisher: Wiley

Date: 1997

DOI: 10.1002/ELPS.1150180320

Abstract: Preparative two‐dimensional polyacrylamide electrophoresis (2‐D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2‐D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious ‘trains’ of spots which differ in p I and/or apparent molecular mass. These are usually isoforms of the same protein and result from post‐translational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes these changes can be visualised on the 2‐D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2‐D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2‐D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2‐D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2‐D gels.

The Dictyostelium discoideum proteome - The SWISS-2DPAGE database of the multicellular aggregate (slug)

Publisher: Wiley

Date: 1997

DOI: 10.1002/ELPS.1150180325

Abstract: The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2‐D PAGE with immobilised pH gradients (pH 3.5–10) in the first dimension and sodium dodecyl sulfate (SDS)‐PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (p I ) and molecular weight ( M r ) against the SWISS‐PROT database with the ExPASy AAcompID tool ( expasy.hcuge.ch/ch2d/aacompi.html ). A total of 25 proteins were identified by matching against database entries for D. discoideum , and another six by crossspecies matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS‐2DPAGE database.

Raw N-glycan mass spectrometry imaging data on formalin-fixed mouse kidney

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.DIB.2018.08.186

UniCarbKB: building a knowledge platform for glycoproteomics

Publisher: Oxford University Press (OUP)

Date: 13-11-2013

DOI: 10.1093/NAR/GKT1128

N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1074/MCP.M116.059816

The glycosylation of human synovial lubricin: implications for its role in inflammation

Publisher: Portland Press Ltd.

Date: 28-06-2010

DOI: 10.1042/BJ20100360

Abstract: Acidic proteins were isolated from synovial fluid from two osteoarthritic and two rheumatoid arthritic patients and identified by MS. It was found that the most abundant protein in all of the s les was the mucin-like protein lubricin. Further characterization of lubricin from the different patients by LC (liquid chromatography)–MS of released oligosaccharides showed that the core 1 O-linked oligosaccharides NeuAcα2–3Galβ1–3GalNAc and NeuAcα2–3Galβ1–3(NeuAcα2–6)GalNAc were the dominating structures on lubricin. The latter was found to be more prevalent in the rheumatoid arthritis s les, indicating that sialylation is up-regulated as part of the inflammatory response. In addition to these dominating structures, core 2 structures were also found in low amounts, where the largest was the disialylated hexasaccharide corresponding to the sequence NeuAcα2–3Galβ1–3(NeuAcα2–3Galβ1–3/4GlcNAcβ1–6)GalNAc. It was also found that a small proportion of the core 2 oligosaccharides carried sulfate. The ability of lubricin to present complex glycosylation reflecting the state of the joint tissue makes lubricin a candidate as a carrier of inflammatory oligosaccharide epitopes. In particular, it was shown that lubricin from inflamed arthritic tissue was recognized by the antibody MECA-79 and thus carried the sulfated epitope proposed to be part of the L-selectin ligand that is responsible for recruitment of leucocytes to inflammatory sites.

Recombinant Prespore‐Specific Antigen from Dictyostelium discoideum is a β‐sheet Glycoprotein with a Spacer Peptide Modified by O‐linked N‐acetylglucosamine

Publisher: Wiley

Date: 06-1996

DOI: 10.1111/J.1432-1033.1996.0511Z.X

Abstract: Prespore-specific antigen (PsA) is a putative cell-adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell-surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl-PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell-surface molecules and here we present information on the structure of the N-terminal globular domain and determine the reducing-terminal linkage of the O-glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of beta-sheet in the N-terminal globular domain, a feature commonly observed in cell-adhesion proteins. Solid-phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O-glycosylations were determined to be single N-acetylglucosamine residues.

Desialylated LDL uptake in human and mouse macrophages can be mediated by a lectin receptor

Publisher: Elsevier BV

Date: 03-1996

DOI: 10.1016/0021-9150(95)05715-3

Abstract: We have compared the uptake of desialylated low density lipoprotein (LDL) with other modified forms of LDL in mouse peritoneal macrophages and PMA-activated human U937 monocytes. Neuraminidase-treated LDL (NT-LDL) caused significant cholesterol ester accumulation in both cell types, although the efficiency relative to loading with acetylated LDL (AcLDL) was markedly different, suggesting a very different complement of receptors in the cells. We therefore determined the effect of PMA-activation on lipoprotein receptor expression in U937 cells and found that while scavenger receptor concentration was elevated after PMA-activation, there was no significant change in the expression of the LDL receptor. Receptor specificity of NT-LDL uptake was examined by competition experiments using the degradation assay. This showed that 125I-labelled NT-LDL uptake in U937 cells could largely be accounted for by the persistent expression of the LDL receptor in these cells. In contrast, in mouse peritoneal macrophages where LDL receptor expression is very low, 125I-labelled NT-LDL degradation was also effectively competed by asialofetuin. Surprisingly, 125I-labelled NT-LDL degradation was also effectively competed by AcLDL. Measurement of sialic acid content of AcLDL showed that approximately 14% of the LDL sialic acid, equivalent to 2 to 3 residues per particle, was lost during acetylation of LDL with acetic anhydride. Thus competition between 125I-labelled NT-LDL and AcLDL could be due to lectin receptor binding rather than competition for scavenger receptor binding.

3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters

Publisher: Springer Science and Business Media LLC

Date: 16-08-2019

DOI: 10.1038/S41467-019-11603-0

Abstract: Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF 4 , doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

Combined N-Glycome and N-Glycoproteome Analysis of the Lotus japonicus Seed Globulin Fraction Shows Conservation of Protein Structure and Glycosylation in Legumes

Publisher: American Chemical Society (ACS)

Date: 25-06-2013

DOI: 10.1021/PR400224S

Abstract: Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial interactions and, due to its well characterized genotype henotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed using mass spectrometry-based glycomics and glycoproteomics techniques. In total, 19 N-glycan structures comprising high mannose (∼20%), pauci-mannosidic (∼40%), and complex forms (∼40%) were determined. The pauci-mannosidic and complex N-glycans contained high amounts of the typical plant determinants β-1,2-xylose and α-1,3-fucose. Two abundant Lotus seed N-glycoproteins were site-specifically profiled a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, Lotus convicilin storage protein 2 (LCP2) carried exclusively high mannose N-glycans similar to its homologue, Ara h 1, which is the major allergen in peanut. In silico investigation confirmed that peanut Ara h 1 and Lotus LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that Lotus has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy.

MALDI mass spectrometry imaging of early- and late-stage serous ovarian cancer tissue reveals stage-specific N-glycans

Publisher: Wiley

Date: 16-08-2019

DOI: 10.1002/PMIC.201800482

Abstract: Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)

Liver disease alters the glycosylation of proteins

Publisher: Wiley

Date: 11-09-2012

DOI: 10.1111/J.1440-1746.2011.07251.X

Proteomics

Publisher: Wiley

Date: 04-2000

DOI: 10.1002/(SICI)1522-2683(20000401)21:6<1071::AID-ELPS1071>3.0.CO;2-M

In-depthN-glycome profiling of paired colorectal cancer and non-tumorigenic tissues reveals cancer-, stage- and EGFR-specific protein N-glycosylation

Publisher: Oxford University Press (OUP)

Date: 17-06-2015

DOI: 10.1093/GLYCOB/CWV042

Abstract: Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-liquid chromatography-tandem mass spectrometry-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-Glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6 ± 13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an overrepresentation of high mannose, hybrid and paucimannosidic type N-glycans and an under-representation of complex N-glycans (P < 0.05). Sialylation, in particular α2,6-sialylation, was significantly higher in CRC tumors relative to non-tumorigenic tissues, whereas α2,3-sialylation was down-regulated (P < 0.05). CRC stage-specific N-glycosylation was detected by high α2,3-sialylation and low bisecting β1,4-GlcNAcylation and Lewis-type fucosylation in mid-late relative to early stage CRC. Interestingly, a novel link between the EGFR status and the N-glycosylation was identified using hierarchical clustering of the N-glycome profiles. EGFR-specific N-glycan signatures included high bisecting β1,4-GlcNAcylation and low α2,3-sialylation (both P < 0.05) relative to EGFR-negative CRC tissues. This is the first study to correlate CRC stage and EGFR status with specific N-glycan features, thus advancing our understanding of the mechanisms causing the biomolecular deregulation associated with CRC.

A Novel Post-translational Modification in Nerve Terminals: O-Linked N-Acetylglucosamine Phosphorylation

Publisher: American Chemical Society (ACS)

Date: 02-05-2011

DOI: 10.1021/PR1011153

Abstract: Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.

Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1

Publisher: Informa UK Limited

Date: 11-01-2018

DOI: 10.1080/14789450.2018.1421946

Abstract: Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories. Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells. Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.

Assessing the role of pharyngeal cell surface glycans in group a streptococcus biofilm formation

Publisher: MDPI AG

Date: 04-11-2020

DOI: 10.3390/ANTIBIOTICS9110775

Abstract: Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20–40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.

Rat Liver Membrane Glycoproteome: Enrichment by Phase Partitioning and Glycoprotein Capture

Publisher: American Chemical Society (ACS)

Date: 06-01-2009

DOI: 10.1021/PR800910W

Abstract: Past proteomic studies of membrane proteins have often been h ered by the low abundance and relatively high hydrophobicity of these proteins. Proteins are often glycosylated, particularly on the extracellular surface of the plasma membrane, and this characteristic was targeted as an enrichment strategy for identifying membrane proteins. Here, we report a strategy for identifying the tissue membrane glycoproteome, which involves (1) Triton X-114 phase partitioning, (2) isolation of glycosylphosphatidylinositol (GPI)-anchored proteins, and (3) glycoprotein capture using lectin affinity or hydrazine chemistry. Surprisingly, the capture of membrane proteins by lectin affinity and hydrazine chemistry resulted in mostly different populations of enriched glycoproteins. Lectins enriched high molecular weight functional membrane proteins with more potential glycosylation such as those involved in signal transduction and cell adhesion. Conversely, hydrazine chemistry isolated a higher proportion of smaller, enzymatic and peripheral membrane proteins such as solute carrier transporters and cytochrome p450s. We have applied our strategy to characterize the rat liver membrane glycoproteome and identified four new predicted GPI-anchored proteins and two that have not previously been seen in the liver. We also identified 424 nonredundant membrane proteins, of which 335 had potential N-linked glycosylation sites.

GlyGen: Computational and Informatics Resources for Glycoscience

Publisher: Oxford University Press (OUP)

Date: 16-10-2019

DOI: 10.1093/GLYCOB/CWZ080

Post-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics

Publisher: American Chemical Society (ACS)

Date: 27-02-2019

DOI: 10.1021/ACS.ANALCHEM.8B05720

Abstract: Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content h ers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc. 2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.

Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni

Publisher: Elsevier BV

Date: 02-2011

DOI: 10.1074/MCP.M000031-MCP201

The 2022 Report on the Human Proteome from the HUPO Human Proteome Project

Publisher: American Chemical Society (ACS)

Date: 11-2022

DOI: 10.1021/ACS.JPROTEOME.2C00498

Abstract: The 2022 Metrics of the Human Proteome from the HUPO Human Proteome Project (HPP) show that protein expression has now been credibly detected (neXtProt PE1 level) for 18 407 (93.2%) of the 19 750 predicted proteins coded in the human genome, a net gain of 50 since 2021 from data sets generated around the world and reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 78 from 1421 to 1343. This represents continuing experimental progress on the human proteome parts list across all the chromosomes, as well as significant reclassifications. Meanwhile, applying proteomics in a vast array of biological and clinical studies continues to yield significant findings and growing integration with other omics platforms. We present highlights from the Chromosome-Centric HPP, Biology and Disease-driven HPP, and HPP Resource Pillars, compare features of mass spectrometry and Olink and Somalogic platforms, note the emergence of translation products from ribosome profiling of small open reading frames, and discuss the launch of the initial HPP Grand Challenge Project, "A Function for Each Protein".

Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52

Publisher: Frontiers Media SA

Date: 27-08-2019

DOI: 10.3389/FIMMU.2019.01967

Molecular analysis of the phospholipids of Escherichia coli K12

Publisher: Elsevier BV

Date: 03-1982

DOI: 10.1016/0005-2760(82)90123-0

Abstract: Phospholipids from Escherichia coli K12 were converted to 1,2-diacylglycerols with phospholipase C from Bacillus cereus. High-pressure liquid chromatography of 1,2-diacylglycerol p-methoxybenzoates on LiChrosorb RP-18 using 2-propanol/acetonitrile (35:65) as eluant permitted separation of 14 molecular species. The main combinations of fatty acids were 1-16:0-2-16:1, 1-16:0-2-cyclo-17:0 and 1-16:0-2-18:1. Positional isomers were not present. The 1,2-di-16:0 compound was present at a significant level (7-10 mol%). Proportions of molecular species varied between phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Phospholipid from the outer membrane of E. coli K12 contained a lower level of molecules with two unsaturated chains than was present in the cytoplasmic membrane. The method is sensitive, has good resolving power and employs readily available equipment.

Genetically and Phenotypically Distinct Pseudomonas aeruginosa Cystic Fibrosis Isolates Share a Core Proteomic Signature

Publisher: Public Library of Science (PLoS)

Date: 02-10-2015

DOI: 10.1371/JOURNAL.PONE.0138527

Negative ion graphitised carbon nano‐liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis

Publisher: Wiley

Date: 07-09-2004

DOI: 10.1002/RCM.1626

Abstract: Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS(2)), using graphitised carbon as separating medium, were explored for analysing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 microL/min) coupled with a conventional electrospray ionisation source, the nano-LC/MS (flow rate of 0.6 microL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The absolute signals for the [M-nH](n-) ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS(2) mode, in which detection of low abundant sequence ions is necessary for characterisation of highly sialylated N-linked oligosaccharides. Oligosaccharides with high numbers of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymatic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS(2) analysis was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitised carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 microg of total amount of protein).

Deletion of the asialyloglycoprotein receptor-1 causes athero-protective effects in vitro and in vivo

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 08-11-2022

DOI: 10.1161/CIRC.146.SUPPL_1.13417

Abstract: Introduction: The asialoglycoprotein receptor-1 (ASGR-1) is known to be a hepatic receptor that clears desialylated glycoproteins from the circulation. A loss-of-function mutation in ASGR-1 was reported to associate with a 34% reduction in coronary artery disease (CAD) risk, suggesting a role for ASGR-1 in CAD. Hypothesis: ASGR-1 is an important player in atherosclerosis and deletion of ASGR-1 will reduce atherosclerotic plaque via athero-protective mechanisms in macrophages. Methods and Results: Using immunofluorescence, we detected ASGR-1 in aortic sinus plaques from apolipoprotein (Apo)e-/- mice fed a high cholesterol diet (HCD)-fed apolipoprotein (Apo)e-/- mice. Bone marrow-derived macrophages (BMDMs) from Asgr1-/- mice elicited greater cholesterol efflux (39%, P .05) and decreased oxLDL uptake (15%, P .05), compared to wildtype BMDMs. Plasma from Asgr-1 -/- mice had lower total cholesterol (25%, P .05) and LDL cholesterol (36%, P .05) concentrations than wildtype control mice. Apoe -/- x Asgr-1 -/+ mice fed HCD for 8 weeks developed less plaque in the aortic sinus (32.75%, P .001) than Apoe -/- controls, and had fewer circulating neutrophils (38%, P .05). In the tandem stenosis model of unstable plaque, Apoe -/- x Asgr-1 -/- mice also developed smaller plaques in their carotid arteries (49%, P .05), than Apoe -/- controls. Furthermore, ASGR-1 was measured in peripheral blood mononuclear cells (PBMCs) isolated from patient s les (n=10-12/group). ASGR-1 protein levels were higher in PBMCs from patients with coronary plaque (61%, P .05), than those without plaque, as determined from coronary angiograms. Conclusions: ASGR-1 plays an important role in atherosclerosis. Deletion of ASGR-1 causes athero-protective effects in macrophages in vitro and reduced plaque burden in vivo . Elevated ASGR-1 levels in PBMCs from clinical blood s les was also associated with the presence of coronary plaque. These studies have significant implications for the potential of ASGR-1 as a therapeutic target for the prevention of atherosclerosis.

Altered N-linked glycosylation in endometrial cancer

Publisher: Springer Science and Business Media LLC

Date: 21-11-2020

DOI: 10.1007/S00216-020-03039-Z

Determination of site-specific glycan heterogeneity on glycoproteins

Publisher: Springer Science and Business Media LLC

Date: 07-06-2012

DOI: 10.1038/NPROT.2012.062

Abstract: The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing ex les of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including s le preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.

GlycoSuiteDB

Publisher: Oxford University Press (OUP)

Date: 2003

DOI: 10.1093/NAR/GKG099

Abstract: GlycoSuiteDB is an annotated and curated relational database of glycan structures reported in the literature. It contains information on the glycan type, core type, linkages and anomeric configurations, mass, composition and the analytical methods used by the researchers to determine the glycan structure. Native and recombinant sources are detailed, including species, tissue and/or cell type, cell line, strain, life stage, disease, and if known the protein to which the glycan structures are attached. There are links to SWISS-PROT/TrEMBL and PubMed where applicable. Recent developments include the implementation of searching by 2D structure and substructure, disease and reference. The database is updated twice a year, and now contains over 7650 entries. Access to GlycoSuiteDB is available at www.glycosuite.com.

Structural analysis of N- and O-glycans released from glycoproteins

Publisher: Springer Science and Business Media LLC

Date: 07-06-2012

DOI: 10.1038/NPROT.2012.063

Abstract: This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The s le preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the s les analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

N-Glycan matrix-assisted laser desorption/ionization mass spectrometry imaging protocol for formalin-fixed paraffin-embedded tissues

Publisher: Wiley

Date: 18-04-2017

DOI: 10.1002/RCM.7845

Abstract: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of the proteome of a tissue has been an established technique for the past decade. In the last few years, MALDI-MSI of the N-glycome has emerged as a novel MALDI-MSI technique. To assess the accuracy and clinical significance of the N-linked glycan spatial distribution, we have developed a method that utilises MALDI-MSI followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) in order to assign glycan structures to the differentiating MALDI-MSI glycan masses released from the tissue glycoproteins. Our workflow presents a comprehensive list of instructions on how to (i) apply MALDI-MSI to spatially map the N-glycome across formalin-fixed paraffin-embedded (FFPE) clinical s les, (ii) structurally characterise N-glycans extracted from consecutive FFPE tissue sections by LC/MS/MS, and (iii) match relevant N-glycan masses from MALDI-MSI with confirmed N-glycan structures determined by LC/MS/MS. Our protocol provides groups that are new to this technique with instructions how to establish N-glycan MALDI-MSI in their laboratory. Furthermore, the method assigns N-glycan structural detail to the masses obtained in the MALDI-MS image. Copyright © 2017 John Wiley & Sons, Ltd.

Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and CLARITY-cleared central nervous system tissues (Conference Presentation)

Publisher: SPIE

Date: 26-04-2016

DOI: 10.1117/12.2209249

Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy

Publisher: Oxford University Press (OUP)

Date: 09-10-2015

DOI: 10.1093/GLYCOB/CWU108

Abstract: As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 μL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between in iduals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR.

Cell surface protein glycosylation in cancer

Publisher: Wiley

Date: 03-2014

DOI: 10.1002/PMIC.201300387

Abstract: Glycosylation of proteins is one of the most important PTMs, with more than half of all human proteins estimated to be glycosylated. It is widely known that aberrant glycosylation has been implicated in many different diseases due to changes associated with biological function and protein folding. In cancer, there is increasing evidence pertaining to the role of glycosylation in tumour formation and metastasis. Alterations in cell surface glycosylation, particularly terminal motifs, can promote invasive behaviour of tumour cells that ultimately lead to the progression of cancer. While a majority of studies have investigated protein glycosylation changes in cancer cell lines and tumour tissue for in idual cancers, the review presented here represents a comprehensive, in-depth overview of literature on the structural changes of glycosylation and their associated synthetic enzymes in five different cancer types originating from the breast, colon, liver, skin and ovary. More importantly, this review focuses on key similarities and differences between these cancers that reflect the importance of structural changes of cell surface N- and O-glycans, such as sialylation, fucosylation, degree of branching and the expression of specific glycosyltransferases for each cancer. It is envisioned that the understanding of these biologically relevant glycan alterations on cellular proteins will facilitate the discovery of novel glycan-based biomarkers which could potentially serve as diagnostic and prognostic indicators of cancer.

Glycoproteome remodeling in MLL-rearranged B-cell precursor acute lymphoblastic leukemia.

Publisher: Ivyspring International Publisher

Date: 2021

DOI: 10.7150/THNO.65398

Liver Membrane Proteome Glycosylation Changes in Mice Bearing an Extra-hepatic Tumor

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1074/MCP.M900538-MCP200

The Year of Glycosciences

Publisher: Oxford University Press (OUP)

Date: 09-01-2013

DOI: 10.1093/GLYCOB/CWS167

Glycomics & Glycoproteomics: From Analytics to Function

Publisher: Royal Society of Chemistry (RSC)

Date: 2021

DOI: 10.1039/D0MO90019B

Abstract: Morten Thaysen-Andersen, Daniel Kolarich and Nicolle H. Packer introduce the Molecular Omics themed issue on Glycomics & Glycoproteomics: From Analytics to Function.

Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product

Publisher: Oxford University Press (OUP)

Date: 03-1999

DOI: 10.1093/GLYCOB/9.3.293

Abstract: The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the in idual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Publisher: Elsevier BV

Date: 2020

DOI: 10.1074/MCP.RA119.001677

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus

Publisher: MDPI AG

Date: 31-05-2019

DOI: 10.3390/MOLECULES24112083

Abstract: We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological s les. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338–BHHTEGST–Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.

Characterization of N- and O-linked glycosylation changes in milk of the tammar wallaby (Macropus eugenii) over lactation

Publisher: Springer Science and Business Media LLC

Date: 10-10-2013

DOI: 10.1007/S10719-012-9452-8

Abstract: As one of several biologically active compounds in milk, glycoproteins have been indicated to be involved in the protection of newborns from bacterial infection. As much of the physical and immune development of the tammar wallaby (Macropus eugenii) young occurs during the early phases of lactation and not in utero, the tammar is a model species for the characterization of potential developmental support agents provided by maternal milk.In the present study, the N- and O-linked glycans from tammar wallaby milk glycoproteins from six in iduals at different lactation time points were subjected to glycomics analyses using porous graphitized carbon liquid chromatography electrospray ionization mass spectrometry. Structural characterization identified a erse range of glycan structures on wallaby milk glycoproteins including sialylated, sulphated, core fucosylated and O-fucosylated structures. 30 % of N-linked structures contained a core (α1-6) fucose. Several of these structures may play roles in development, and exhibit statistically significant temporal changes over the lactation period. The N-glycome was found to contain structures with NeuGc residues, while in contrast the O-glycome did not. O-fucosylated structures were identified in the early stages of lactation indicating a potential role in the early stages of development of the pouch young. Overall the results suggest that wallaby milk contains structures known to have developmental and immunological significance in human milk and reproduction in other animals, highlighting the importance of glycoproteins in milk.

The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets

Publisher: Oxford University Press (OUP)

Date: 09-2016

DOI: 10.1093/GLYCOB/CWW082

Abstract: The minimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and s le preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for s le preparation. The s le preparation guidelines include all aspects of s le generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE s le preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets.

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1074/MCP.O115.057638

Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells

Publisher: Elsevier BV

Date: 11-2011

DOI: 10.1074/MCP.M111.009001

Advances in LC–MS/MS-based glycoproteomics: Getting closer to system-wide site-specific mapping of the N- and O-glycoproteome

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBAPAP.2014.05.002

Abstract: Site-specific structural characterization of glycoproteins is important for understanding the exact functional relevance of protein glycosylation. Resulting partly from the multiple layers of structural complexity of the attached glycans, the system-wide site-specific characterization of protein glycosylation, defined as glycoproteomics, is still far from trivial leaving the N- and O-linked glycoproteomes significantly under-defined. However, recent years have seen significant advances in glycoproteomics driven, in part, by the developments of dedicated workflows and efficient s le preparation, including glycopeptide enrichment and prefractionation. In addition, glycoproteomics has benefitted from the continuous performance enhancement and more intelligent use of liquid chromatography and tandem mass spectrometry (LC-MS/MS) instrumentation and a wider selection of specialized software tackling the unique challenges of glycoproteomics data. Together these advances promise more streamlined N- and O-linked glycoproteome analysis. Tangible ex les include system-wide glycoproteomics studies detecting thousands of intact glycopeptides from hundreds of glycoproteins from erse biological s les. With a strict focus on the system-wide site-specific analysis of protein N- and O-linked glycosylation, we review the recent advances in LC-MS/MS based glycoproteomics. The review opens with a more general discussion of experimental designs in glycoproteomics and s le preparation prior to LC-MS/MS based data acquisition. Although many challenges still remain, it becomes clear that glycoproteomics, one of the last frontiers in proteomics, is gradually maturing enabling a wider spectrum of researchers to access this new emerging research discipline. The next milestone in analytical glycobiology is being reached allowing the glycoscientist to address the functional importance of protein glycosylation in a system-wide yet protein-specific manner.

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.EXER.2016.01.013

Abstract: The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with α2-3,6,8 neuraminidase (sialidase) and α1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye.

Discrimination of Isomers of Released N- and O-Glycans Using Diagnostic Product Ions in Negative Ion PGC-LC-ESI-MS/MS

Publisher: American Chemical Society (ACS)

Date: 30-03-2018

DOI: 10.1007/S13361-018-1932-Z

Abstract: Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play erse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers. Graphical Abstract ᅟ.

Terminal galactosylation and sialylation switching on membrane glycoproteins upon TNF-alpha-induced insulin resistance in adipocytes

Publisher: Elsevier BV

Date: 2016

DOI: 10.1074/MCP.M115.054221

Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS

Publisher: Wiley

Date: 12-2011

DOI: 10.1002/ELPS.201100294

Abstract: Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour-associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glycopeptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation.

Comparison of the methods for profiling glycoprotein glycans—HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study

Publisher: Oxford University Press (OUP)

Date: 12-01-2007

DOI: 10.1093/GLYCOB/CWL086

Abstract: Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the ergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same s les of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these s les. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

There Are No Facts, Only Interpretations

Publisher: American Chemical Society (ACS)

Date: 06-2006

DOI: 10.1021/PR0627286

UniCarbKB: An Emergent Knowledge Base for Glycomics

Publisher: Springer Japan

Date: 2014

DOI: 10.1007/978-4-431-54836-2_21-1

High-performance liquid chromatography of diglyceride p-nitrobenzoates

Publisher: Elsevier BV

Date: 10-1980

DOI: 10.1016/S0021-9673(00)80524-5

Abstract: Inflammatory contributions from diet and adiposity may interact with respect to the development of type 2 diabetes mellitus (T2DM). We investigated the degree to which adiposity modified the association between dietary inflammatory potential and incident T2DM. Data from 6,016 US men in the Aerobics Center Longitudinal Study who completed a 3-day diet record were used. The inflammatory potential of diet was characterized by the Dietary Inflammatory Index (DII®), and adiposity was assessed with body mass index, waist circumference, body fat percentage (BF) and waist-to-height ratio. Inverse probability weights were used in modified Poisson regression models to examine whether adiposity modifies the relationship between the DII and T2DM, while accounting for selection bias from participants who were lost to follow-up. There were 336 incident cases of T2DM after a mean follow-up of 6.5 years. DII scores were not significantly associated with T2DM incidence in multivariable models, but point estimates were consistently elevated across increasing DII quartiles compared to the most anti-inflammatory DII quartile. In the model that evaluated BF, the term for overall effect modification was significant (p = 0.02), but there was no evidence of effect modification on the multiplicative and additive scales when examined further. Effect modification was not present for any other adiposity measures. We did not observe evidence that a pro-inflammatory diet, as measured by the DII, is associated with incidence of T2DM, nor evidence that adiposity modifies a potential relationship. Further investigation is needed in larger cohorts with longer follow-up.

A continuous culture method for obtaining 100% triangular cells of Trigonopsis variabilis

Publisher: Microbiology Society

Date: 08-1978

DOI: 10.1099/00221287-107-2-377

Total Synthesis of Homogeneous Antifreeze Glycopeptides and Glycoproteins

Publisher: Wiley

Date: 02-03-2012

DOI: 10.1002/ANIE.201108682

Interaction between Polysialic Acid and the MARCKS-ED Peptide at the Molecular Level

Publisher: American Chemical Society (ACS)

Date: 15-05-2020

DOI: 10.1021/ACSCHEMNEURO.0C00139

An Interactive View of Glycosylation

Publisher: Springer US

Date: 06-10-2021

DOI: 10.1007/978-1-0716-1685-7_3

Abstract: The present chapter focuses on the interactive and explorative aspects of bioinformatics resources that have been recently released in glycobiology. The comparative analysis of data in a field where knowledge is scattered, incomplete, and disconnected from main biology requires efficient visualization, integration, and interactive tools that are currently only partially implemented. This overview highlights converging efforts toward building a consistent picture of protein glycosylation.

Site-Specific Glycan-Peptide Analysis for Determination of N-Glycoproteome Heterogeneity

Publisher: American Chemical Society (ACS)

Date: 11-2013

DOI: 10.1021/PR400783J

Abstract: A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.

Structural analysis of glycoprotein sialylation – part II: LC-MS based detection

Publisher: Royal Society of Chemistry (RSC)

Date: 2013

DOI: 10.1039/C3RA42969E

GlycoDigest: a tool for the targeted use of exoglycosidase digestions in glycan structure determination

Publisher: Oxford University Press (OUP)

Date: 10-07-2014

DOI: 10.1093/BIOINFORMATICS/BTU425

Abstract: Summary: Sequencing oligosaccharides by exoglycosidases, either sequentially or in an array format, is a powerful tool to unambiguously determine the structure of complex N- and O- link glycans. Here, we introduce GlycoDigest, a tool that simulates exoglycosidase digestion, based on controlled rules acquired from expert knowledge and experimental evidence available in GlycoBase. The tool allows the targeted design of glycosidase enzyme mixtures by allowing researchers to model the action of exoglycosidases, thereby validating and improving the efficiency and accuracy of glycan analysis. Availability and implementation: www.glycodigest.org . Contact: matthew.c bell@mq.edu.au or frederique.lisacek@isb-sib.ch

UniCarbKB

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.BBAGEN.2016.02.016

Abstract: UniCarbKB aims to provide a resource for the representation of mammalian glycobiology knowledge by providing a curated database of structural and experimental data, supported by a web application that allows users to easily find and view richly annotated information. The database comprises two levels of annotation (i) global-specific data of oligosaccharides released and characterised from single purified glycoproteins and (ii) information pertaining to site-specific glycan heterogeneity. Additional, contextual information is provided including structural, bibliographic, and taxonomic information for each entry. Since the launch of UniCarbKB in 2012, we have continued to improve the organisation of our data model. Recently, we have extended our pipeline to collate structural and abundance changes of oligosaccharides in different human disease states and experimental models to extend our coverage of the human glycome. In this manuscript, we demonstrate the capability of UniCarbKB to store and query relative glycan abundance data using a set of published colorectal and prostate cancer cell lines as ex les. Furthermore, we outline our strategy for managing large-scale glycoproteomics data, site-specific and glycan compositional data, and how this information is adding value to UniCarbKB. Finally, we summarise our efforts to improve the efficient representation of disease terms and associated changes in glycan heterogeneity by integrating the Disease Ontology. Updates and improvements to UniCarbKB have introduced unique features for storing and displaying glycosylation features of mammalian glycoproteins. The integration of site-specific glycosylation data obtained from large-scale glycoproteomics and introduction of cell line studies will improve the analysis of glycoproteins and entire glycomes. Continuing advancements in analytical technologies and new data types are advancing disease-related glycomics. It is increasingly necessary to ensure all the data are comprehensively annotated. UniCarbKB was established with the mission of providing a resource for human glycobiology by capturing a wide range of data with corresponding annotations. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1074/MCP.M110.006833

Lipopolysaccharide and Morphine-3-Glucuronide-Induced Immune Signalling Increases the Expression of Polysialic Acid in PC12 Cells

Publisher: Springer Science and Business Media LLC

Date: 24-10-2020

DOI: 10.1007/S12035-019-01791-7

Abstract: Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.

Glycosylation of sputum mucins is altered in cystic fibrosis patients

Publisher: Oxford University Press (OUP)

Date: 28-03-2007

DOI: 10.1093/GLYCOB/CWM036

Abstract: Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after s ling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.

Final Program: 59th ASMS Conference on Mass Spectrometry and Allied Topics, June 5-9, 2011, Denver, Colorado

Publisher: American Chemical Society (ACS)

Date: 30-03-2011

DOI: 10.1007/S13361-011-0127-7

Simple Urinary Sample Preparation for Proteomic Analysis

Publisher: American Chemical Society (ACS)

Date: 06-09-2006

DOI: 10.1021/PR060305Y

Abstract: Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal s le preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.

Erratum

Publisher: Oxford University Press (OUP)

Date: 05-2007

DOI: 10.1093/GLYCOB/CWM041

33 Linking protein N-glycosylation and CF: malfunctioning CFTR and pulmonary infection yield aberrant N-glycosylation of sputum derived proteins of CF individuals

Publisher: Elsevier BV

Date: 06-2014

DOI: 10.1016/S1569-1993(14)60170-3

One gene, many proteins

Publisher: Springer Berlin Heidelberg

Date: 2007

DOI: 10.1007/978-3-540-72910-5_5

Facile Assembly of Functional Upconversion Nanoparticles for Targeted Cancer Imaging and Photodynamic Therapy

Publisher: American Chemical Society (ACS)

Date: 06-05-2016

DOI: 10.1021/ACSAMI.6B00713

Abstract: The treatment depth of existing photodynamic therapy (PDT) is limited because of the absorption of visible excitation light in biological tissue. It can be augmented by means of upconversion nanoparticles (UCNPs) transforming deep-penetrating near-infrared (NIR) light to visible light, exciting PDT drugs. We report here a facile strategy to assemble such PDT nanocomposites functionalized for cancer targeting, based on coating of the UCNPs with a silica layer encapsulating the Rose Bengal photosensitizer and bioconjugation to antibodies through a bifunctional fusion protein consisting of a solid-binding peptide linker genetically fused to Streptococcus Protein G'. The fusion protein (Linker-Protein G) mediates the functionalization of silica-coated UCNPs with cancer cell antibodies, allowing for specific target recognition and delivery. The resulting nanocomposites were shown to target cancer cells specifically, generate intracellular reactive oxygen species under 980 nm excitation, and induce NIR-triggered phototoxicity to suppress cancer cell growth in vitro.

Symbol Nomenclature for Graphical Representations of Glycans

Publisher: Oxford University Press (OUP)

Date: 05-11-2015

DOI: 10.1093/GLYCOB/CWV091

GlycoMod - A software tool for determining glycosylation compositions from mass spectrometric data

Publisher: Wiley

Date: 02-2001

DOI: 10.1002/1615-9861(200102)1:2<340::AID-PROT340>3.0.CO;2-B

Structural glycobiology: Applications in organ transplantation

Publisher: CRC Press

Date: 27-09-2012

DOI: 10.1201/B12965

GlycoSuiteDB

Publisher: Oxford University Press (OUP)

Date: 2001

DOI: 10.1093/NAR/29.1.332

Abstract: GlycoSuiteDB is a relational database that curates information from the scientific literature on glyco-protein derived glycan structures, their biological sources, the references in which the glycan was described and the methods used to determine the glycan structure. To date, the database includes most published O:-linked oligosaccharides from the last 50 years and most N:-linked oligosaccharides that were published in the 1990s. For each structure, information is available concerning the glycan type, linkage and anomeric configuration, mass and composition. Detailed information is also provided on native and recombinant sources, including tissue and/or cell type, cell line, strain and disease state. Where known, the proteins to which the glycan structures are attached are reported, and cross-references to the SWISS-PROT/TrEMBL protein sequence databases are given if applicable. The GlycoSuiteDB annotations include literature references which are linked to PubMed, and detailed information on the methods used to determine each glycan structure are noted to help the user assess the quality of the structural assignment. GlycoSuiteDB has a user-friendly web interface which allows the researcher to query the database using mono-isotopic or average mass, monosaccharide composition, glycosylation linkages (e.g. N:- or O:-linked), reducing terminal sugar, attached protein, taxonomy, tissue or cell type and GlycoSuiteDB accession number. Advanced queries using combinations of these parameters are also possible. GlycoSuiteDB can be accessed on the web at www.glycosuite.com.

Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O

Publisher: American Chemical Society (ACS)

Date: 25-08-2014

DOI: 10.1021/PR5005554

Abstract: C ylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.

Bright upconversion nanoparticles under light-emitting diode excitation

Publisher: SPIE

Date: 30-12-2019

DOI: 10.1117/12.2541246

Letter to the glycoforum transforming glycoscience: An Australian perspective

Publisher: Oxford University Press (OUP)

Date: 06-12-2014

DOI: 10.1093/GLYCOB/CWT103

Toward Automated N-Glycopeptide Identification in Glycoproteomics

Publisher: American Chemical Society (ACS)

Date: 02-09-2016

DOI: 10.1021/ACS.JPROTEOME.6B00438

Abstract: Advances in software-driven glycopeptide identification have facilitated N-glycoproteomics studies reporting thousands of intact N-glycopeptides, i.e., N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we compare the site-specific glycoprofiling efficiency of the PTM-centric search engine Byonic relative to manual expert annotation utilizing typical glycoproteomics acquisition and data analysis strategies but with a single glycoprotein, the uncharacterized multiple N-glycosylated human basigin. Detailed site-specific reference glycoprofiles of purified basigin were manually established using ion-trap CID-MS/MS and high-resolution Q-Exactive Orbitrap HCD-MS/MS of tryptic N-glycopeptides and released N-glycans. The micro- and macroheterogeneous basigin N-glycosylation was site-specifically glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS. The automated glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target/decoy proteome databases. Within the limits of this single glycoprotein analysis, the search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the automated glycoprofiling efficiency and analysis time. Correct identification of ambiguous peptide modifications (methionine oxidation/carbamidomethylation) whose mass differences coincide with several monosaccharide mass differences (Fuc/Hex/HexNAc) and of ambiguous isobaric (Hex

Utilization of a Dextran Ladder to Standardize PGC-LC-MS-Based Glycomics

Publisher: American Chemical Society (ACS)

Date: 12-03-2018

DOI: 10.26434/CHEMRXIV.7056767

Abstract: Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or s le-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological s les is evidenced by the ability to discriminate between cell culture and tissue s le types by the normalized intensity of i N- /i glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation. br

Challenges of determining o-glycopeptide heterogeneity

Publisher: American Chemical Society (ACS)

Date: 13-04-2010

DOI: 10.1021/AC901717N

Abstract: O-Linked glycosylation often occurs in mucin-type domains that are heavily and heterogeneously glycosylated and are challenging to analyze. The analysis of these domains is often overlooked because of these difficulties, but changes in mucinlike domain glycosylation are implicated in many diseases. Here we have explored several strategies to determine the heterogeneity of mucinlike O-glycosylated domains. Four glucanases secreted in large quantities from Trichoderma reesei, all containing heavily O-glycosylated mucinlike linker regions, were used as a model system. The strategies involved monosaccharide compositional analysis and identification of the released glycans by HPAEC-PAD and carbon-LC ESI-MS/MS. Glycosylated peptides were generated by different protease digestions (trypsin, papain, Asp-N, PreTAQ) and enriched by HILIC microcolumns, to determine the glycopeptide heterogeneity and glycosylation sites. The complex O-glycan heterogeneity on the intact glycoproteins and the enriched mucin-type domains was determined by MALDI-MS and ESI-MS, but the dense O-glycosylation in the mucin-type domains conferred high resistance to protease cleavage. ETD-MS/MS of the glycopeptide-enriched protease digests was unsuccessful for the de novo assignment of O-glycosylation at in idual sites within the mucin-type domains but allowed several previously unknown O-linked sites outside the defined linker region to be found on two of the four glucanases. The protease digests produced many glycopeptides as determined by CID-MS/MS, but ETD fragmentation of these resulted in only a few interpretable spectra, suggesting that the use of ETD for determining the heterogeneous O-glycosylation at specific sites in regions of multiple occupancy is still in its infancy.

Utilising Glycobiology for Fluorescent Nanodiamond Uptake and Imaging in the Central Nervous System

Publisher: IEEE

Date: 06-2019

DOI: 10.1109/PIERS-SPRING46901.2019.9017296

Glycosylation of acetylxylan esterase from Trichoderma reesei

Publisher: Oxford University Press (OUP)

Date: 04-2002

DOI: 10.1093/GLYCOB/12.4.291

Abstract: The nature of the N- and O- linked glycosylation of acetylxylan esterase (AXE) of the Trichoderma reesei strain Rut-C30 has been characterized using different enzymatic, chromatographic, and mass spectrometric techniques. The combined data showed that the AXE N-glycan is phosphorylated and highly mannosylated. The predominant N-glycans on the single glycosylation site on AXE can be represented as GlcNAc(2)Man((1-6))P. The linker-substrate binding domain peptide separated from the core by papain digestion is heavily O-glycosylated and consists of mannose, galactose, and possibly glucose as monosaccharide and disaccharide substituents. In addition to glycosylation, sulfation was observed in the linker region. Both N- and O- linked glycans show remarkable heterogeneity. Three isoforms of AXE, separated by 2D SDS-PAGE, are described with pI values of 5.0, 5.3, and 5.9. The three isoforms can be explained by posttranslational modification of the enzyme by glycans, phosphate, and sulfate. Advancing the knowledge on the nature of the glycans produced by T. reesei is elementary for its use as a host for the expression of heterologous glycoproteins of industrial and pharmaceutical importance.

Glycoproteomics of Milk: Differences in Sugar Epitopes on Human and Bovine Milk Fat Globule Membranes

Publisher: American Chemical Society (ACS)

Date: 15-07-2008

DOI: 10.1021/PR700793K

Abstract: Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.

Mouse brain glycomics – Insights from exploring the Allen Brain Atlas and the implications for the neuroimmune brain

Publisher: Elsevier BV

Date: 10-2023

DOI: 10.1016/J.BBI.2023.06.024

Translating N-glycan analytical applications into clinical strategies for ovarian cancer

Publisher: Wiley

Date: 09-11-2019

DOI: 10.1002/PRCA.201800099

Abstract: Protein glycosylation, particularly N-linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell-cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS-based techniques, to qualitatively and quantitatively assess N-glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC-ESI-MS/MS and MALDI time-of-flight MS (MALDI-TOF-MS), which have been used to analyze clinical s les, such as serum, plasma, ascites, and tissue. Targeting the aberrant N-glycosylation patterns observed in MALDI-MS imaging (MSI) offers a platform to visualize N-glycans in tissue-specific regions. The studies on the intra-patient (i.e., a comparison of tissue-specific regions from the same patient) and inter-patient (i.e., a comparison of tissue-specific regions between different patients) variation of early- and late-stage ovarian cancer (OC) patients identify specific N-glycan differences that improve understanding of the tumor microenvironment and potentially improve therapeutic strategies for the clinic.

GlycoViewer

Publisher: Oxford University Press (OUP)

Date: 25-05-2010

DOI: 10.1093/NAR/GKQ446

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

Publisher: Wiley

Date: 16-10-2019

DOI: 10.1002/PMIC.201900010

Abstract: While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man

GlyConnect: Glycoproteomics Goes Visual, Interactive, and Analytical

Publisher: American Chemical Society (ACS)

Date: 21-12-2018

DOI: 10.1021/ACS.JPROTEOME.8B00766

Abstract: Knowledge of glycoproteins, their site-specific glycosylation patterns, and the glycan structures that they present to their recognition partners in health and disease is gradually being built on using a range of experimental approaches. The data from these analyses are increasingly being standardized and presented in various sources, from supplemental tables in publications to localized servers in investigator laboratories. Bioinformatics tools are now needed to collect these data and enable the user to search, display, and connect glycomics and glycoproteomics to other sources of related proteomics, genomics, and interactomics information. We here introduce GlyConnect ( glyconnect.expasy.org/ ), the central platform of the Glycomics@ExPASy portal for glycoinformatics. GlyConnect has been developed to gather, monitor, integrate, and visualize data in a user-friendly way to facilitate the interpretation of collected glycoscience data. GlyConnect is designed to accommodate and integrate multiple data types as they are increasingly produced.

Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1074/MCP.M113.037085

Incorporation of 5‐aminolevulinic acid into chlorophyll in darkness in barley

Publisher: Wiley

Date: 10-1986

DOI: 10.1111/J.1399-3054.1986.TB01918.X

A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells.

Publisher: Wiley

Date: 16-03-2015

DOI: 10.1002/RCM.7130

Abstract: Glycosphingolipids (GSLs) constitute a highly erse class of glyco-conjugates which are involved in many aspects of cell membrane function and disease. The isolation, detection and structural characterization of the carbohydrate (glycan) component of GSLs are particularly challenging given their structural heterogeneity and thus rely on the development of sensitive, analytical technologies. Neutral and acidic GSL standards were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymatically released using endoglycoceramidase II (EGCase II), separated by porous graphitized carbon (PGC) liquid chromatography and structurally characterized by negative ion mode electrospray ionization tandem mass spectrometry (PGC-LC/ESI-MS/MS). This approach was then employed for GSLs isolated from 100 mg of serous and endometrioid cancer tissue and from cell line (10(7) cells) s les. Glycans were released from GSL standards comprising of ganglio-, asialo-ganglio- and the relatively resistant globo-series glycans, using as little as 1 mU of enzyme and 2 µg of GSL. The platform of analysis was then applied to GSLs isolated from tissue and cell line s les and the released isomeric and isobaric glycan structures were chromatographically resolved on PGC and characterized by comparison with the MS(2) fragment ion spectra of the glycan standards and by application of known structural MS(2) fragment ions. This approach identified several (neo-)lacto-, globo- and ganglio-series glycans and facilitated the discrimination of isomeric structures containing Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides. We describe a relatively simple, detergent-free, enzymatic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural analysis afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the analysis of tumour and cell-derived GSL-glycans. The method uses the potential of MS(2) fragmentation in negative ion ESI mode to characterize, in detail, the biologically relevant glycan structures derived from GSLs.

Detection of rare prostate cancer cells in human urine offers prospect of non-invasive diagnosis

Publisher: Springer Science and Business Media LLC

Date: 02-11-2022

DOI: 10.1038/S41598-022-21656-9

Abstract: Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4–10 cells/mL. Blinded analysis of each patient urine s le, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3–20 PCa cells per mL by TGiA, 4–13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine s les of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa.

The Lectin Riddle: Glycoproteins Fractionated from Complex Mixtures Have Similar Glycomic Profiles

Publisher: Mary Ann Liebert Inc

Date: 08-2010

DOI: 10.1089/OMI.2010.0075

Abstract: One common method used for analyzing the glycoproteome is chromatography using multiple lectins that display different affinities toward oligosaccharide structures. Much has been done to determine lectin affinity using standard glycoproteins with known glycosylation however, a knowledge of the selectivity and specificity of lectins exposed to complex mixtures of proteins is required if they are to be used as a means of studying the glycoproteome. In the present study, three lectins (Concanavalin A, Jacalin, and Wheat Germ Agglutinin) were used to fractionate glycoproteins from two different complex environments: (1) cell membranes and (2) plasma. Reproducible enrichment of glycoproteins from these s les has been shown to result from the combined use of these lectins. However, the global glycan profiles of the released N- and O-linked oligosaccharides from the glycoproteins retained by the lectins, and from those glycoproteins that did not bind, using both these complex s les, were found to be very similar. That is, although the lectins selectively and reproducibly retained some glycoproteins, other proteins with the same attached oligosaccharide structures did not bind. Some small N- and O-glycan differences were observed in the bound fractions but there was little absolute specificity toward in idual oligosaccharide structures known to have high affinity to these lectins. These data indicate that lectins are useful for fractionating glycoproteins from complex mixtures, but that the overall glycoproteome is not isolated by this approach.

Macquarie University

Location: Australia

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Griffith University Institute For Glycomics

Location: Australia

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Super Science Fellowships - Grant ID: FS110200026

Start Date: 12-2011

End Date: 06-2015

Amount: $556,800.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1094624

Start Date: 2010

End Date: 04-2013

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100204

Start Date: 2016

End Date: 12-2016

Amount: $550,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100130

Start Date: 09-2010

End Date: 11-2011

Amount: $850,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110104958

Start Date: 01-2011

End Date: 12-2014

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100101

Start Date: 03-2011

End Date: 12-2012

Amount: $160,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC130100009

Start Date: 05-2013

End Date: 09-2017

Amount: $2,100,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE130100007

Start Date: 2013

End Date: 12-2014

Amount: $650,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0455407

Start Date: 07-2004

End Date: 12-2008

Amount: $389,997.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200102004

Start Date: 05-2020

End Date: 05-2023

Amount: $411,079.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE140100003

Start Date: 10-2014

End Date: 12-2020

Amount: $23,000,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE140100111

Start Date: 2014

End Date: 06-2016

Amount: $475,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE220100157

Start Date: 04-2022

End Date: 12-2023

Amount: $535,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP130100517

Start Date: 09-2013

End Date: 09-2016

Amount: $427,510.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100038

Start Date: 01-2012

End Date: 06-2014

Amount: $654,000.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE200100029

Start Date: 11-2020

End Date: 11-2027

Amount: $35,000,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC210100040

Start Date: 02-2022

End Date: 02-2027

Amount: $4,997,903.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100150

Start Date: 2010

End Date: 12-2012

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882855

Start Date: 2008

End Date: 12-2009

Amount: $900,000.00

Funder: Australian Research Council