ORCID Profile
0000-0001-8327-6843
Current Organisations
Nagoya University
,
Macquarie University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Bioprocessing, Bioproduction and Bioproducts | Biochemistry and Cell Biology | Medicinal and Biomolecular Chemistry | Analytical Biochemistry | Biologically Active Molecules
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Chemical Sciences | Nutraceuticals and Functional foods | Expanding Knowledge in the Biological Sciences | Service Industries Standards and Calibrations |
Related Links
Publications
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.
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.
Bioanalytical evaluation of dried plasma spots for monitoring of abiraterone and ∆(4)-abiraterone from cancer patients
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.JCHROMB.2019.121741
Abstract: Abiraterone acetate is an approved prodrug administered orally in a fixed dose format for the treatment of metastatic castration-resistant prostate cancer (mCRPC). In vivo, the prodrug is readily metabolized to abiraterone and its active metabolite Δ(4)-abiraterone (D4A) which selectively and irreversibly inhibit the 17α-hydroxylase/17,20-lyase (CYP17A1) enzyme and the androgen receptor, respectively. Therapeutic drug monitoring (TDM) of abiraterone and its metabolites may be beneficial as significant pharmacokinetic variability has been observed. Dried plasma spots (DPS) represent an attractive, yet under-utilised approach for TDM analysis with desired features including easy collection, transport, storage and overcomes the issues of blood hematocrit levels known in dried blood spot analysis. In this study we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of abiraterone and D4A with deuterated internal standard (abiraterone D4) from DPS using a high-resolution benchtop mass spectrometer. Calibration curves were linear over a wide and clinically-relevant concentration range (0.132-196.0 ng/mL for abiraterone and 0.110-39.17 ng/mL for D4A) with high accuracy (93-104% for abiraterone and 96-108% for D4A) and precision (%CV ≤ 12.5). As expected, the levels of abiraterone and D4A obtained from DPS from mCRPC patients varied substantially (1.5-31.4 ng/mL for abiraterone and 0.1-5.2 ng/mL for D4A n = 22). Detailed benchmarking of the DPS method to a pre-validated liquid plasma method showed that the techniques generate quantitative indistinguishable data. Collectively, this demonstrates the potential of using LC-MS/MS in combination with DPS for TDM of abiraterone and D4A from patients.
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
Human protein paucimannosylation: cues from the eukaryotic kingdoms
Publisher: Wiley
Date: 11-02-2202
DOI: 10.1111/BRV.12548
Abstract: Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α- or β-mannosyl-terminating asparagine (N)-linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue- and subcellular-specific expression of PMPs within invertebrates and plants, often comprising multi-α1,3/6-fucosylation and β1,2-xylosylation amongst other glycan modifications and non-glycan substitutions e.g. O-methylation. Vertebrates and protists express less-heterogeneous PMPs typically only comprising variable core fucosylation of bi- and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less-truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in erse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue-wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell-cell communication and host-pathogen/symbiont interactions. In most PMP-producing organisms, including humans, the N-acetyl-β-hexosaminidase isoenzymes and linkage-specific α-mannosidases are glycoside hydrolases critical for generating PMPs via N-acetylglucosaminyltransferase I (GnT-I)-dependent and GnT-I-independent truncation pathways. However, the identity and structure of many species-specific PMPs in eukaryotes, their biosynthetic routes, strong tissue- and development-specific expression, and erse functions are still elusive. Deep exploration of these PMP features involving, for ex le, the characterisation of endogenous PMP-recognising lectins across a variety of healthy and N-acetyl-β-hexosaminidase-deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongst the many tasks required for enhanced insight into the glycobiology of human PMPs. In conclusion, the literature supports the notion that PMPs are significant, yet still heavily under-studied biomolecules in human glycobiology that serve essential functions and create structural heterogeneity not dissimilar to other human N-glycoprotein types. Human PMPs should therefore be recognised as bioactive glycoproteins that are distinctly different from the canonical N-glycoprotein classes and which warrant a more dedicated focus in glycobiological research.
Comprehensive Glycoprofiling of Oral Tumors Associates N-Glycosylation With Lymph Node Metastasis and Patient Survival
Publisher: Elsevier BV
Date: 07-2023
Comprehensive glycoprofiling of oral tumours associatesN-glycosylation with lymph node metastasis and patient survival
Publisher: Cold Spring Harbor Laboratory
Date: 22-11-2022
DOI: 10.1101/2022.11.21.517331
Abstract: While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), its heterogeneous glycoproteome and dynamics with disease progression remain unmapped. To this end, we here employ an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics applied to a valuable cohort of resected tumour tissues from OSCC patients with (n = 19) and without (n = 12) lymph node metastasis. While all tumour tissues displayed uniform N -glycome profiles suggesting relatively stable global N -glycosylation during lymph node metastasis, glycoproteomics and advanced correlation analysis notably uncovered altered site-specific N -glycosylation and previously unknown associations with several key clinicopathological features. Importantly, focused analyses of the multi-omics data unveiled two N -glycans and three N -glycopeptides that were closely associated with patient survival. This study provides novel insight into the complex OSCC tissue N -glycoproteome forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glyco-markers for OSCC. Deep survey of the dynamic landscape of complex sugars in oral tumours paves a way for new prognostic disease markers.
Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay
Publisher: Elsevier BV
Date: 09-2021
Structural basis for the recognition of nectin-like protein-5 by the human-activating immune receptor, DNAM-1
Publisher: Elsevier BV
Date: 08-2019
Serum N-Glycomics Stratifies Bacteremic Patients Infected with Different Pathogens
Publisher: MDPI AG
Date: 02-2021
DOI: 10.3390/JCM10030516
Abstract: Bacteremia—i.e., the presence of pathogens in the blood stream—is associated with long-term morbidity and is a potential precursor condition to life-threatening sepsis. Timely detection of bacteremia is therefore critical to reduce patient mortality, but existing methods lack precision, speed, and sensitivity to effectively stratify bacteremic patients. Herein, we tested the potential of quantitative serum N-glycomics performed using porous graphitized carbon liquid chromatography tandem mass spectrometry to stratify bacteremic patients infected with Escherichia coli (n = 11), Staphylococcus aureus (n = 11), Pseudomonas aeruginosa (n = 5), and Streptococcus viridans (n = 5) from healthy donors (n = 39). In total, 62 N-glycan isomers spanning 41 glycan compositions primarily comprising complex-type core fucosylated, bisecting N-acetylglucosamine (GlcNAc), and α2,3-/α2,6-sialylated structures were profiled across all s les using label-free quantitation. Excitingly, unsupervised hierarchical clustering and principal component analysis of the serum N-glycome data accurately separated the patient groups. P. aeruginosa-infected patients displayed prominent N-glycome aberrations involving elevated levels of fucosylation and bisecting GlcNAcylation and reduced sialylation relative to other bacteremic patients. Notably, receiver operating characteristic analyses demonstrated that a single N-glycan isomer could effectively stratify each of the four bacteremic patient groups from the healthy donors (area under the curve 0.93–1.00). Thus, the serum N-glycome represents a new hitherto unexplored class of potential diagnostic markers for bloodstream infections.
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
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
Comprehensive glycoprofiling of the epimastigote and trypomastigote stages of Trypanosoma cruzi
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.JPROT.2016.05.034
Abstract: Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving insect and mammalian hosts and distinct developmental stages. During T. cruzi developmental stages, glycoproteins play important role in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. In this study, comprehensive glycoprofiling analysis was performed in the epimastigote and trypomastigote stages of T. cruzi using two glycopeptide enrichment strategies, lectin-based and hydrophilic interaction liquid chromatography, followed by high resolution LC-MS/MS. Following deglycosylation, a total of 1306 N-glycosylation sites in NxS/T/C motifs were identified from 690 T. cruzi glycoproteins. Among them, 170 and 334 glycoproteins were exclusively identified in epimastigotes and trypomastigotes, respectively. Besides, global site-specific characterization of the N- and O-linked glycan heterogeneity in the two life stages of T. cruzi was achieved by intact glycopeptide analysis, revealing 144/466 unique N-linked and 10/97 unique O-linked intact glycopeptides in epimastigotes/trypomastigotes, respectively. Conclusively, this study documents the significant T. cruzi stage-specific expression of glycoproteins that can help to better understand the T. cruzi phenotype and response caused by the interaction with different hosts during its complex life cycle. Chagas disease caused by the protozoan Trypanosoma cruzi is a neglected disease which affects millions of people especially in Latin America. The absence of efficient drugs and vaccines against Chagas disease stimulates the search for novel targets. Glycoproteins are very attractive therapeutic candidate targets since they mediate key processes in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. This study aimed to provide an in depth characterization of the N-linked and O-linked glycoproteome of two T. cruzi life stages: epimastigotes and trypomastigotes. Mass spectrometry-based proteomics showed interesting stage-specific glycoproteome signatures that are valuable to better understand the importance of protein glycosylation in epimastigotes and trypomastigotes and to expand the repertoire of potential therapeutic targets against Chagas disease.
Relative versus absolute quantitation in disease glycomics
Publisher: Wiley
Date: 24-03-2015
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.
Investigation of the detoxification mechanism of formaldehyde-treated tetanus toxin
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.VACCINE.2006.12.033
Abstract: The tetanus vaccine is based on the extremely potent tetanus neurotoxin (TeNT), which is converted by treatment with formaldehyde and lysine into the non-toxic, but still immunogenic tetanus toxoid (TTd). This formaldehyde-induced detoxification, which to a large extend determines the quality and properties of the vaccine component, occurs through partly unknown chemical modifications of the toxin. The aim of this study was to gain knowledge of the detoxification mechanism in the generation of the tetanus vaccine. Two approaches were chosen: (i) the effect of changes in the concentrations of lysine and formaldehyde in the detoxification process and (ii) characterisation of the chemically detoxified TTd. (i) We examined a number of TTd components that was produced by varying the concentrations of formaldehyde and lysine during the inactivation. Toxicity tests showed that the detoxification failed when the lysine or formaldehyde concentration was < or =1/5 or < or =1/10, respectively, of the standard level. Gel-electrophoretic analyses showed that inter-chain cross-linking was formaldehyde-dependent and, furthermore, revealed that inter-chain cross-linking was not the only requirement for the inactivation. In addition, the measurable amount of tyrosine correlated inversely with the degree of inter-chain cross-linking. (ii) To study the formaldehyde-induced chemical modifications, the TTd was investigated using protein chemical techniques in combination with mass spectrometry (MS). Using off-line liquid chromatography (LC)-MS, the most pronounced chemical modifications were characterised as unstable Schiff-bases (+12 Da) located on lysine residues and the N-termini of peptides throughout the molecule. Several arginine residues were also found with +12 Da modifications due to Schiff-base formation or as a consequence of degenerative fragmentation of lysine/formaldehyde adducts or cross-links during MS. A few tyrosine residues were similarly observed with a mass increase of 12 Da. Even though it cannot be ruled out that this is a residual mass of higher molecule adducts or cross-links to tyrosine, amino acid analysis and MS data indicated that the modification forms a ring structure from a carbon in the aromatic ring to the backbone N(alpha). In addition, several mono-epsilon-methyllysines (+14 Da) were observed as a likely consequence of reductive methylation of the Schiff-bases. A substantial part (87%) of the known TeNT sequence, including the active site, was covered using the off-line LC-MS approach to investigate the tryptic digested TTd. In contrast to the results obtained from the gel-electrophoretic experiments, neither intra/inter-chain cross-links nor cross-links to external lysines were observed in the MS analysis. Instability of the cross-links during separation and/or MS is likely to explain their absence in the analyses. The biological relevance of the observed modifications is discussed in relation to 3D mapping analyses. Proposals for the TeNT detoxification are discussed, although no direct evidence for the exact mechanism could be obtained.
Macrophage-derived secretome is sufficient to confer olanzapine-mediated insulin resistance in human adipocytes
Publisher: Elsevier BV
Date: 08-2021
The Function of the Human Interferon-β1a Glycan Determined in Vivo
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 29-04-2008
Abstract: Recombinant human interferon-beta (rhIFN-beta) is the leading therapeutic intervention shown to change the cause of relapsing-remitting multiple sclerosis, and both a nonglycosylated and a significantly more active glycosylated variant of rhIFN-beta are used in treatment. This study investigates the function of the rhIFN-beta1a glycan moiety and its in idual carbohydrate residues, using the myxovirus resistance (Mx) mRNA as a biomarker in Mx-congenic mice. We showed that the Mx mRNA level in blood leukocytes peaked 3 h after s.c. administration of rhIFN-beta1a. In addition, a clear dose-response relationship was confirmed, and the Mx response was shown to be receptor-mediated. Using specific glycosidases, different glycosylation analogs of rhIFN-beta1a were obtained, and their activities were determined. The glycosylated rhIFN-beta1a showed significantly higher activity than its deglycosylated counterpart, due to a protein stabilization/solubilization effect of the glycan. It is interesting to note that the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-beta1a was determined in vivo, and it provided a novel insight into the role of the rhIFN-beta1a glycan and its carbohydrate residues. The possibilities of improving the pharmacological properties of rhIFN-beta1a using glycoengineering are discussed.
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
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.
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
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.
Oligomannosylation and MAN1A1 expression associate strongly with a subset of human cancer types
Publisher: Cold Spring Harbor Laboratory
Date: 09-05-2021
DOI: 10.1101/2021.05.08.443254
Abstract: Aberrant protein glycosylation is a prominent cancer feature. While many tumour-associated glycoepitopes have been reported, advances in glycoanalytics continue to uncover new associations between glycoproteins and cancer. Guided by a comprehensive literature survey suggesting that oligomannosylation (Man 5-9 GlcNAc 2 , M5-M9) is a widespread albeit poorly studied glyco-signature in human cancers, we here re-visit a valuable compilation of nearly 500 LC-MS/MS N -glycomics datasets acquired across 11 human cancer types to systematically test for oligomannose-cancer associations. Firstly, our quantitative glycomics data obtained across 34 cancerous cell lines demonstrated that oligomannosylation, particularly the under-processed M7-M9, is a strong pan-cancer feature. We then showed cell surface expression of oligomannosidic epitopes in the promyelocytic leukemic HL-60 cell line using concanavalin A-based flow cytometry. In keeping with literature, our quantitative glycomics data of tumour and matching control tissues and new MALDI-MS imaging data of tissue microarrays showed a strong cancer-associated elevation of oligomannosylation in both basal cell ( p = 1.78 x 10 -12 ) and squamous cell ( p = 1.23 x 10 -11 ) skin cancer and colorectal cancer ( p = 8.0 x 10 -4 ). The glycomics data also indicated that few cancer types including gastric and liver cancer exhibit unchanged or reduced oligomannose levels, observations also supported by literature and MALDI-MSI. Finally, data from cancer repositories indicated that three α1,2-mannosidases dictate oligomannose expression in cancer cells, and further suggested that deleterious mutations and reduced expression of MAN1A1 are key contributors to the cancer-associated oligomannose elevation. Collectively, these findings open hitherto unexplored avenues for the development of new cancer biomarkers and therapeutic targets.
Cystic fibrosis and bacterial colonization define the sputum N-glycosylation phenotype
Publisher: Oxford University Press (OUP)
Date: 04-09-2014
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.
Investigating the biomarker potential of glycoproteins using comparative glycoprofiling — application to tissue inhibitor of metalloproteinases-1
Publisher: Elsevier BV
Date: 03-2008
DOI: 10.1016/J.BBAPAP.2007.12.007
Abstract: Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted s les using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the s le origin. This study clearly illustrates that the technique is capable of performing in idualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins.
Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum
Publisher: Elsevier BV
Date: 04-2015
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.
Analysis of Protein Glycosylation and Phosphorylation Using HILIC-MS
Publisher: CRC Press
Date: 17-02-2011
DOI: 10.1201/B10609-24
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
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 interactions of calreticulin with immunoglobulin G and immunoglobulin Y
Publisher: Elsevier BV
Date: 07-2011
DOI: 10.1016/J.BBAPAP.2011.03.015
Abstract: Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.
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.
The Complexity and Dynamics of the Tissue Glycoproteome Associated With Prostate Cancer Progression
Publisher: Elsevier BV
Date: 2021
Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development
Publisher: Elsevier BV
Date: 2021
Identifying sources and estimating glandular output of salivary TIMP‐1
Publisher: Informa UK Limited
Date: 2008
Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions*.
Publisher: Elsevier BV
Date: 08-2017
Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface
Publisher: American Chemical Society (ACS)
Date: 13-06-2014
DOI: 10.1021/AC5010212
Structural analysis of glycoprotein sialylation – Part I: pre-LC-MS analytical strategies
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3RA42960A
N-Glycans Modulate the Function of Human Corticosteroid-Binding Globulin
Publisher: Elsevier BV
Date: 08-2011
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.
Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)
Publisher: Springer Science and Business Media LLC
Date: 05-05-2009
Abstract: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.
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
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.
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.
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.
Critical considerations in N-glycoproteomics
Publisher: Elsevier BV
Date: 04-2023
2D gels still have a niche in proteomics
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.JPROT.2013.01.010
Abstract: With the rapid advance of MS-based proteomics one might think that 2D gel-based proteomics is dead. This is far from the truth. Current research has shown that there are still a number of places in the field of protein and molecular biology where 2D gels still play a leading role. The aim of this review is to highlight some of these applications. Ex les from our own research as well as from other published works are used to illustrate the 2D gel driven research in the areas of: 1) de novo sequencing and protein identification from organisms with no or incomplete genome sequences available 2) alternative detection methods for modification specific proteomics 3) identification of protein isoforms and modified proteins. With an ex le of the glycoprotein TIMP-1 protein we illustrate the unique properties of 2D gels for the separation and characterisation of multiply modified proteins. We also show that careful analysis of experimental and theoretical protein mass and pI can lead to the identification of unanticipated protein variants modified by for ex le proteolytic cleavage. Together this shows that there is an important niche for 2D gel-based proteomics, which compliments traditional LC-MS techniques for specific protein research purposes.
Trends in oligomannosylation and α1,2-mannosidase expression in human cancers
Publisher: Impact Journals, LLC
Date: 12-10-2021
Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells
Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.PEP.2014.06.013
Abstract: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.
N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils
Publisher: Oxford University Press (OUP)
Date: 18-10-2021
Abstract: We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0–1), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilization of a putative noncanonical truncation pathway in favor of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB−/−) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate PMP formation via a putative noncanonical truncation pathway in neutrophils.
Characterization of sialylated and fucosylated glycopeptides of β2‐glycoprotein I by a combination of HILIC LC and MALDI MS/MS
Publisher: Wiley
Date: 03-2010
Abstract: Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N ‐glycans at each in idual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter‐ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human β2‐glycoprotein I (β2‐GPI, apolipoprotein H), which contains multiple N ‐glycosylation sites. Conditions for rapid proteolytic digestion of β2‐GPI using low‐specificity proteases were optimized to detect β2‐GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi‐ and tri‐antennary complex type glycans, were characterized at three N ‐glycosylation sites, namely Asn‐143, Asn‐174 and Asn‐234, of β2‐GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein s les in functional proteomic research and advance our understanding of the biological role of protein glycosylation.
Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia
Publisher: Wiley
Date: 20-10-2019
Abstract: The histology-based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor s ling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label-free LC-MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from in iduals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate-derived proteins including IGKV3D-20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same s le cohort validate the differential expression of LMOD1, GYG1, IGKV3D-20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness.
Glycomics-Assisted Glycoproteomics Enables Deep and Unbiased N-Glycoproteome Profiling of Complex Biological Specimens
Publisher: Springer US
Date: 2023
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.
Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52
Publisher: Frontiers Media SA
Date: 27-08-2019
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
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
High-resolution longitudinal N- and O-glycoprofiling of human monocyte-to-macrophage transition
Publisher: Oxford University Press (OUP)
Date: 09-03-2020
Abstract: Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon–liquid chromatography–tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal s ling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0–1, 22.1–30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8–35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6–39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.
FUT1genetic variants impact protein glycosylation of porcine intestinal mucosa
Publisher: Oxford University Press (OUP)
Date: 07-02-2016
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.
Glycoproteome remodeling and organelle-specific N -glycosylation accompany neutrophil granulopoiesis
Publisher: Proceedings of the National Academy of Sciences
Date: 28-08-2023
Abstract: Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N -glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow–derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N -glycans low in α2,6-sialylation and Lewis fucosylation decorating a erse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N -glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N -glycoproteome featuring intriguing organelle-specific N -glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors.
Rapid and Individual-specific Glycoprofiling of the Low Abundance N-Glycosylated Protein Tissue Inhibitor of Metalloproteinases-1
Publisher: Elsevier BV
Date: 04-2007
Determination of protein conformation by isotopically labelled cross-linking and dedicated software: Application to the chaperone, calreticulin
Publisher: Elsevier BV
Date: 12-2007
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.
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.
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
Utilizing Ion-Pairing Hydrophilic Interaction Chromatography Solid Phase Extraction for Efficient Glycopeptide Enrichment in Glycoproteomics
Publisher: American Chemical Society (ACS)
Date: 10-06-2010
DOI: 10.1021/AC100530W
Abstract: Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and nonglycosylated peptides. Three mobile phases were investigated, i.e., 2% formic acid (defined as IP(2% FA) ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP(0.1% TFA) and IP(1% TFA) ZIC-HILIC SPE) all containing 80% acetonitrile. S les of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evaluated by enhanced glycopeptide detection using MALDI-TOF MS and RP-LC-ESI-MS/MS, e.g., the glycopeptide ion signals were increased by up to 3.7-fold compared to IP(2% FA) conditions. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 nonglycosylated peptides observed in the IP(2% FA) ZIC-HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopeptide enrichment efficiency considerably for a broad range of s le complexities when using ZIC-HILIC SPE.
Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease
Publisher: Elsevier BV
Date: 06-2016
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.
Program and Abstracts for 2016 Annual Meeting of the Society for Glycobiology
Publisher: Oxford University Press (OUP)
Date: 31-10-2016
Pyrexia and acidosis act independently of neutrophil elastase reactive center loop cleavage to effect cortisol release from corticosteroid‐binding globulin
Publisher: Wiley
Date: 04-11-2020
DOI: 10.1002/PRO.3982
Towards structure-focused glycoproteomics
Publisher: Portland Press Ltd.
Date: 13-01-2021
DOI: 10.1042/BST20200222
Abstract: Facilitated by advances in the separation sciences, mass spectrometry and informatics, glycoproteomics, the analysis of intact glycopeptides at scale, has recently matured enabling new insights into the complex glycoproteome. While erse quantitative glycoproteomics strategies capable of mapping monosaccharide compositions of N- and O-linked glycans to discrete sites of proteins within complex biological mixtures with considerable sensitivity, quantitative accuracy and coverage have become available, developments supporting the advancement of structure-focused glycoproteomics, a recognised frontier in the field, have emerged. Technologies capable of providing site-specific information of the glycan fine structures in a glycoproteome-wide context are indeed necessary to address many pending questions in glycobiology. In this review, we firstly survey the latest glycoproteomics studies published in 2018–2020, their approaches and their findings, and then summarise important technological innovations in structure-focused glycoproteomics. Our review illustrates that while the O-glycoproteome remains comparably under-explored despite the emergence of new O-glycan-selective mucinases and other innovative tools aiding O-glycoproteome profiling, quantitative glycoproteomics is increasingly used to profile the N-glycoproteome to tackle erse biological questions. Excitingly, new strategies compatible with structure-focused glycoproteomics including novel chemoenzymatic labelling, enrichment, separation, and mass spectrometry-based detection methods are rapidly emerging revealing glycan fine structural details including bisecting GlcNAcylation, core and antenna fucosylation, and sialyl-linkage information with protein site resolution. Glycoproteomics has clearly become a mainstay within the glycosciences that continues to reach a broader community. It transpires that structure-focused glycoproteomics holds a considerable potential to aid our understanding of systems glycobiology and unlock secrets of the glycoproteome in the immediate future.
Site-Specific Glycoprofiling of N-Linked Glycopeptides Using MALDI-TOF MS: Strong Correlation between Signal Strength and Glycoform Quantities
Publisher: American Chemical Society (ACS)
Date: 09-04-2009
DOI: 10.1021/AC900231W
Abstract: Site-specific glycoprofiling of N-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, but its quantitative accuracy lacks documentation. Thus, a systematic study of widely different glycopeptides was performed to determine the relationship between the relative abundances of the in idual glycoforms and the MALDI-TOF MS signal strength. Glycopeptides derived from glycoproteins containing neutral glycans (ribonuclease B, IgG, and ovalbumin) were initially profiled and yielded excellent and reproducible quantitation (correlation coefficient r = 0.9958, n = 5) when evaluated against a normal phase HPLC 2-AB glycan profile. Similarly, precise quantitation was observed for various forms of N-glycans (free, permethylated, and fluorescence-labeled) using MS. In addition, three different sialoglycopeptides from fetuin were site-specifically profiled, and good correlation between peak intensities and relative abundances was found with only a minor loss of sialic acids (r = 0.9664, n = 5). For glycopeptide purification, a range of hydrophilic and graphite materials packed in microcolumn format proved capable of performing desalting without loss of quantitative information, but highlighted the column capacity as a critical parameter. In conclusion, MALDI-TOF MS signal strength of glycopeptides has been found to accurately reflect the relative quantities of glycoforms, providing that certain technical issues are considered, i.e., nonbiased s le handling, matrix choice, and instrumental settings. This enables rapid and sensitive site-specific glycoprofiling of N-glycan populations to promote biomarker discovery and elucidation of glycan structure/function relationships.
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.
Paucimannosidic glycoepitopes are functionally involved in proliferation of neural progenitor cells in the subventricular zone
Publisher: Oxford University Press (OUP)
Date: 28-04-2015
Abstract: Protein glycosylation has received much attention due to its multiple functional roles in physiological and pathophysiological conditions. Paucimannose is a common mannosidic N-glycoepitope in invertebrates and plants but has only recently been detected in vertebrates. Herein, we demonstrate the presence of paucimannosidic epitopes specifically in early postnatal neural progenitor cells (NPCs) between postnatal day 0 and 7 in mouse brain suggesting a possible role in the development of NPCs. Paucimannosidic epitopes were also detected in human glioblastoma cells and human macrophages by immunofluorescence and mass spectrometric analysis. Its expression was significantly increased after proliferation arrest indicating its importance in the regulation of cell proliferation. This hypothesis was further strengthened by reduced cell proliferation after the application of paucimannose-reactive Mannitou antibody into culture medium of growing cells. Most interestingly, this reduction in cell proliferation upon the administration of Mannitou antibody could also be observed in vivo in the subventricular zone of early postnatal mouse brain. Taken together, these observations demonstrate that paucimannosylation directly influences cell proliferation in various vertebrate cell types including early postnatal neural stem cells.
Glycoengineered hepatitis B virus-like particles with enhanced immunogenicity
Publisher: Elsevier BV
Date: 05-2020
Glycoproteomics
Publisher: Springer Science and Business Media LLC
Date: 23-06-2022
Terminal galactosylation and sialylation switching on membrane glycoproteins upon TNF-alpha-induced insulin resistance in adipocytes
Publisher: Elsevier BV
Date: 2016
N-Glycosylation influences human corticosteroid-binding globulin measurements
Publisher: Bioscientifica
Date: 08-2019
DOI: 10.1530/EC-19-0242
Abstract: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood s les. We have previously been unable to verify this biochemically, and sought to determine if N -glycosylation differences account for discrepancies in ELISA measurements of CBG. Molecular biological, biochemical and glycopeptide analyses were used to examine how N -glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as ex les of N -glycosylation defects. We demonstrate that an N -glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N -glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N -glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Plasma CBG measurements are influenced by variations in N -glycosylation. This is important given the increasing number of CDG defects identified recently and because N -glycosylation abnormalities are common in patients with metabolic and liver diseases.
Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS
Publisher: Wiley
Date: 12-2011
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.
Total Synthesis of Homogeneous Antifreeze Glycopeptides and Glycoproteins
Publisher: Wiley
Date: 02-03-2012
Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase
Publisher: Cold Spring Harbor Laboratory
Date: 25-07-2020
DOI: 10.1101/2020.07.24.219956
Abstract: Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, structural modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity. Myeloperoxidase (MPO) is an important microbicidal glycoprotein critical for fighting pathogens. We report, for the first time, the intriguingly complex relationship between glycobiology and MPO immune function by demonstrating that uncommon and strategically positioned hyper-truncated glycans both elevate the activity and the inhibition potential of this pathogen-combating enzyme. We have used a multifaceted approach employing integrated biomolecular analytics to generate new insights into the sugar code of MPO. The findings described in this study improve our understanding of key innate immune processes and may guide future glycoengineering efforts aiming to generate therapeutically relevant recombinant MPO products with tuneable activity and inhibition potential tailored to biomedical applications involving persisting and severe pathogen infections.
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.
Glycoproteome remodelling and granule-specificN-glycosylation accompany neutrophil granulopoiesis
Publisher: Cold Spring Harbor Laboratory
Date: 20-01-2023
DOI: 10.1101/2023.01.18.524318
Abstract: Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we perform comprehensive spatiotemporal N -glycoproteome profiling of isolated granule populations from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Interestingly, the granules of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, peculiar highly truncated N -glycosylation in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven primarily by changes in protein expression and less by the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N -glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with high glycosylation efficiencies of the azurophilic granule proteins. Our study provides spatiotemporal insights into the complex neutrophil N -glycoproteome featuring an intriguing granule-specific N -glycosylation formed by dynamic remodelling during myeloid progenitor-to-neutrophil maturation. Systems glycobiology reveals that profound N -glycoproteome remodelling accompanies early neutrophil granulopoiesis Precision glycoproteomics produces detailed cartography of neutrophils that exhibit site-, protein- and granule-specific N-glycosylation
Structural analysis of glycoprotein sialylation – part II: LC-MS based detection
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3RA42969E
Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery
Publisher: Elsevier BV
Date: 09-2020
Structural and functional diversity of neutrophil glycosylation in innate immunity and related disorders
Publisher: Elsevier BV
Date: 09-2021
Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells
Publisher: Elsevier BV
Date: 05-2021
Synthesis of homogeneous MUC1 oligomers via a bi-directional ligation strategy
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3OB41363B
Abstract: The efficient synthesis of homogeneous MUC1 peptide oligomers using sequential ligation reactions in the N-to-C and C-to-N directions is reported. The bi-directional ligation strategy makes use of thioester formation via N → S acyl shift chemistry in combination with peptide ligation reactions and was used to prepare a library of peptide oligomers ranging in molecular mass from 3.8-9.4 kDa, comprised of between 2 and 5 repeats of the MUC1 variable number tandem repeat sequence.
N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research
Publisher: Wiley
Date: 22-05-2017
DOI: 10.1111/TPJ.13570
Abstract: Studies of protein N-glycosylation are important for answering fundamental questions on the erse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the in idual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For ex le, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features.
Interaction of the Chaperone Calreticulin with Proteins and Peptides of Different Structural Classes
Publisher: Bentham Science Publishers Ltd.
Date: 11-2009
DOI: 10.2174/092986609789353772
Abstract: The interaction of calreticulin with native and denatured forms and polypeptides in proteolytic digests of proteins representing structural classes of all-alpha-helix (hemoglobin, serum albumin), all-beta-sheet (IgG) and alpha-helix + beta-sheets (lysozyme, ovalbumin) was investigated. The binding of calreticulin to denatured proteins was found to depend on conformation and structural class of the protein. No interaction was observed with the native proteins, whereas binding was seen for the denatured proteins, the order of interaction being lysozyme = IgG > ovalbumin >> hemoglobin = serum albumin. Moreover, the interaction between calreticulin and the heat-denatured proteins depended on the temperature and time used for denaturation and the degree of proteolytic fragmentation. Calreticulin bound well to peptides in proteolytic digests from protease K or chymotrypsin treatment of lysozyme, IgG and ovalbumin but weakly or not at all to peptides in proteolytic digests of hemoglobin and serum albumin. Synthetic peptides from lysozyme and ovalbumin confirmed binding to hydrophobic peptides from these proteins. These results show that calreticulin has the ability to interact with denatured and fragmented forms of proteins with a preference for beta-strand structure and hydrophobicity.
Mycobacterium tuberculosis Infection Manipulates the Glycosylation Machinery and the N-Glycoproteome of Human Macrophages and Their Microparticles
Publisher: American Chemical Society (ACS)
Date: 04-11-2017
DOI: 10.1021/ACS.JPROTEOME.6B00685
Abstract: Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of frontline alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M. tb infection. Infection promoted a dramatic regulation of the macrophage proteome. Most notably, significant infection-dependent down-regulation (4-26 fold) of 11 lysosomal exoglycosidases, e.g., β-galactosidase, β-hexosaminidases and α-/β-mannosidases, was observed. Relative weak infection-driven transcriptional regulation of these exoglycosidases and a stronger augmentation of the extracellular hexosaminidase activity demonstrated that the lysosome-centric changes may originate predominantly from infection-induced secretion of the lysosomal content. The macrophages showed heterogeneous N-glycan profiles and displayed significant up-regulation of complex-type glycosylation and concomitant down-regulation of paucimannosylation upon infection. Complementary intact N-glycopeptide analysis supported a subcellular-specific manipulation of the glycosylation machinery and altered glycosylation patterns of lysosomal N-glycoproteins within infected macrophages. Interestingly, the corresponding macrophage-derived MPs displayed unique N-glycome and proteome signatures supporting a preferential packaging from plasma membranes. The MPs were devoid of infection-dependent N-glycosylation signatures, but interestingly displayed increased levels of the glyco-initiating oligosaccharyltransferase complex and associated α-glucosidases that correlated with increased formation, N-glycan precursor levels and N-glycan density of infected MPs. In conclusion, this system-wide study provides new insight into the host- and pathogen-driven N-glycoproteome manipulation of macrophages in TB.
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
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
A pain-causing and paralytic ant venom glycopeptide
Publisher: Elsevier BV
Date: 10-2021
Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase
Publisher: Elsevier BV
Date: 2021
Distinct urinary glycoprotein signatures in prostate cancer patients
Publisher: Impact Journals, LLC
Date: 04-09-2018
Paucimannosidic glycoepitopes inhibit tumorigenic processes in glioblastoma multiforme
Publisher: Impact Journals, LLC
Date: 08-07-2019
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.
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
A Defined α-Helix in the BifunctionalO-Glycosylated Natriuretic Peptide TcNPa from the Venom ofTropidechis carinatus
Publisher: Wiley
Date: 03-03-2015
Abstract: Natriuretic peptides (NP) play important roles in human cardiac physiology through their guanylyl cyclase receptors NPR-A and NPR-B. Described herein is a bifunctional O-glycosylated natriuretic peptide, TcNPa, from Tropidechis carinatus venom and it unusually targets both NPR-A and NPR-B. Characterization using specific glycosidases and ETD-MS identified the glycan as galactosyl-β(1-3)-N-acetylgalactosamine (Gal-GalNAc) and was α-linked to the C-terminal threonine residue. TcNPa contains the characteristic NP 17-membered disulfide ring with conserved phenylalanine and arginine residues. Both glycosylated and nonglycosylated forms were synthesized by Fmoc solid-phase peptide synthesis and NMR analysis identified an α-helix within the disulfide ring containing the putative pharmacophore for NPR-A. Surprisingly, both forms activated NPR-A and NPR-B and were relatively resistant towards proteolytic degradation in plasma. This work will underpin the future development of bifunctional NP peptide mimetics.
Synthesis and immunological evaluation of self-adjuvanting MUC1-macrophage activating lipopeptide 2 conjugate vaccine candidates
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4CC03510K
Abstract: The synthesis and immunological evaluation of self-adjuvanting cancer vaccine candidates comprising of mucin 1 (MUC1) (glyco)peptides linked to macrophage activating lipopeptide 2 (MALP2) is described.
Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers
Publisher: Wiley
Date: 16-10-2019
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
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.
Salivary tissue inhibitor of metalloproteinases-1 localization and glycosylation profile analysis
Publisher: Wiley
Date: 22-08-2011
DOI: 10.1111/J.1600-0463.2011.02796.X
Abstract: Salivary tissue inhibitor of metalloproteinases-1 (TIMP-1) has been associated with pathological conditions in the oral cavity, but the origin of TIMP-1 in saliva remains unknown. Hence, we studied the localization of TIMP-1 in salivary gland tissue and also investigated if TIMP-1 found in blood and saliva is identical. Human salivary gland tissue s les (four parotid gland and four submandibular gland biopsies) were analysed for the presence of TIMP-1 mRNA and protein expression. To assess TIMP-1 glycosylation profiles in blood and saliva, the protein was isolated from plasma and unstimulated and stimulated whole saliva as well as stimulated parotid and submandibular saliva and analysed by MALDI-TOF mass spectrometry. TIMP-1 protein was demonstrated in mucous acinar cells of the submandibular gland and in ductal cells of both the parotid and submandibular gland. However, no TIMP-1 mRNA was detected in any of these cells. The glycosylation profiles of TIMP-1 isolated from whole saliva and saliva from the major glands were highly similar. In contrast, a significant difference was found between the glycoprofiles of salivary TIMP-1 and plasma TIMP-1. Although no clear evidence of TIMP-1 transcription in major salivary glands was demonstrated our results suggest that TIMP-1 in saliva does not originate from plasma.
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