ORCID Profile
0000-0002-4823-7758
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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)
Biochemistry and Cell Biology | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Analytical Chemistry | Characterisation of Biological Macromolecules | Microbiology | Analytical Spectrometry | Nanochemistry and Supramolecular Chemistry | Manufacturing Engineering not elsewhere classified | Horticultural Production | Proteins and Peptides | Receptors and Membrane Biology | Enzymes | Microbial Genetics | Medical Biotechnology | Ecological Impacts of Climate Change | Horticultural Crop Growth and Development | Medical Molecular Engineering of Nucleic Acids and Proteins | Analytical Biochemistry | Cell Metabolism | Bacteriology
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Infectious Diseases | Ornamentals, Natives, Flowers and Nursery Plants | Crop Protection Chemicals | Manufacturing not elsewhere classified | Human Pharmaceutical Treatments (e.g. Antibiotics) | Expanding Knowledge in the Chemical Sciences | Immune System and Allergy |
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Publications
Saliva proteome research: current status and future outlook
Publisher: Informa UK Limited
Date: 21-05-2013
DOI: 10.3109/07388551.2012.687361
Abstract: Human saliva harbours proteins of clinical relevance and about 30% of blood proteins are also present in saliva. This highlights that saliva can be used for clinical applications just as urine or blood. However, the translation of salivary biomarker discoveries into clinical settings is h ered by the dynamics and complexity of the salivary proteome. This review focuses on the current status of technological developments and achievements relating to approaches for unravelling the human salivary proteome. We discuss the dynamics of the salivary proteome, as well as the importance of s le preparation and processing techniques and their influence on downstream protein applications post-translational modifications of salivary proteome and protein: protein interactions. In addition, we describe possible enrichment strategies for discerning post-translational modifications of salivary proteins, the potential utility of selected-reaction-monitoring techniques for biomarker discovery and validation, limitations to proteomics and the biomarker challenge and future perspectives. In summary, we provide recommendations for practical saliva s ling, processing and storage conditions to increase the quality of future studies in an emerging field of saliva clinical proteomics. We propose that the advent of technologies allowing sensitive and high throughput proteome-wide analyses, coupled to well-controlled study design, will allow saliva to enter clinical practice as an alternative to blood-based methods due to its simplistic nature of s ling, non-invasiveness, easy of collection and multiple collections by untrained professionals and cost-effective advantages.
The intrinsic and regulated proteomes of barley seeds in response to fungal infection
Publisher: Cold Spring Harbor Laboratory
Date: 18-10-2018
DOI: 10.1101/445916
Abstract: Barley is an important cereal grain used for beer brewing, animal feed, and human food consumption. Fungal disease can impact barley production, as it causes substantial yield loss and lowers seed quality. We used sequential window acquisition of all theoretical ions mass spectrometry (SWATH-MS) to measure and quantify the relative abundance of proteins within seeds of different barley varieties under various fungal pathogen burdens (ProteomeXchange Datasets PXD011303 and PXD014093). Fungal burden in the leaves and stems of barley resulted in changes to the seed proteome. However, these changes were minimal and showed substantial variation among barley s les infected with different pathogens. The limited effect of intrinsic disease resistance on the seed proteome is consistent with the main mediators of disease resistance being present in the leaves and stems of the plant. The seeds of barley varieties accredited for use as malt had higher levels of proteins associated with starch synthesis and beer quality. The proteomic workflows developed and implemented here have potential application in quality control, breeding and processing of barley, and other agricultural products.
Sirtuins in the phylum Basidiomycota: A role in virulence in Cryptococcus neoformans
Publisher: Springer Science and Business Media LLC
Date: 21-04-2017
DOI: 10.1038/SREP46567
Abstract: Virulence of Cryptococcus neoformans is regulated by a range of transcription factors, and is also influenced by the acquisition of adaptive mutations during infection. Beyond the temporal regulation of virulence factor production by transcription factors and these permanent microevolutionary changes, heritable epigenetic modifications such as histone deacetylation may also play a role during infection. Here we describe the first comprehensive analysis of the sirtuin class of NAD+ dependent histone deacetylases in the phylum Basidiomycota, identifying five sirtuins encoded in the C. neoformans genome. Each sirtuin gene was deleted and a wide range of phenotypic tests performed to gain insight into the potential roles they play. Given the pleiotropic nature of sirtuins in other species, it was surprising that only two of the five deletion strains revealed mutant phenotypes in vitro . However, cryptic consequences of the loss of each sirtuin were identified through whole cell proteomics, and mouse infections revealed a role in virulence for SIR2, HST3 and HST4 . The most intriguing phenotype was the repeated inability to complement mutant phenotypes through the reintroduction of the wild-type gene. These data support the model that regulation of sirtuin activity may be employed to enable a drastic alteration of the epigenetic landscape and virulence of C. neoformans .
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.
Convergent microevolution of Cryptococcus neoformans hypervirulence in the laboratory and the clinic
Publisher: Springer Science and Business Media LLC
Date: 20-12-2017
DOI: 10.1038/S41598-017-18106-2
Abstract: Reference strains are a key component of laboratory research, providing a common background allowing for comparisons across a community of researchers. However, laboratory passage of these strains has been shown to lead to reduced fitness and the attenuation of virulence in some species. In this study we show the opposite in the fungal pathogen Cryptococcus neoformans , with analysis of a collection of type strain H99 subcultures revealing that the most commonly used laboratory subcultures belong to a mutant lineage of the type strain that is hypervirulent. The pleiotropic mutant phenotypes in this H99L (for “Laboratory”) lineage are the result of a deletion in the gene encoding the SAGA Associated Factor Sgf29, a mutation that is also present in the widely-used H99L-derived KN99 a /α congenic pair. At a molecular level, loss of this gene results in a reduction in histone H3K9 acetylation. Remarkably, analysis of clinical isolates identified loss of function SGF29 mutations in C. neoformans strains infecting two of fourteen patients, demonstrating not only the first ex le of hypervirulence in clinical C. neoformans s les, but also parallels between in vitro and in vivo microevolution for hypervirulence in this important pathogen.
The pilin O-glycosylation pathway of pathogenic Neisseria is a general system that glycosylates AniA, an outer membrane nitrite reductase
Publisher: Elsevier BV
Date: 2009
DOI: 10.1016/J.BBRC.2008.11.025
Abstract: O-Glycosylation is emerging as a common posttranslational modification of surface exposed proteins in bacterial mucosal pathogens. In pathogenic Neisseria an O-glycosylation pathway modifies a single abundant protein, pilin, the subunit protein that forms pili. Here, we identify an additional outer membrane glycoprotein in pathogenic Neisseria, the nitrite reductase AniA, that is glycosylated in its C-terminal repeat region by the pilin glycosylation pathway. To our knowledge, this is the first report of a general O-glycosylation pathway in a prokaryote. We also show that AniA displays polymorphisms in residues that map to the surface of the protein. A frame-shift mutation abolishes AniA expression in 34% of Neisseria meningitidis strains surveyed, however, all Neisseria gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology.
Glycoproteomic measurement of site-specific polysialylation
Publisher: Cold Spring Harbor Laboratory
Date: 20-08-2019
DOI: 10.1101/740928
Abstract: Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings.
Group a Streptococcus M1T1 intracellular infection of primary tonsil epithelial cells dampens levels of secreted IL-8 through the action of SpyCEP
Publisher: Frontiers Media SA
Date: 17-05-2018
MUC5B glycosylation in human saliva reflects blood group and secretor status
Publisher: Oxford University Press (OUP)
Date: 06-04-2005
Abstract: This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from in iduals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose olyacrylamide composite gel electrophoresis (SDS-AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive beta-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O in iduals into larger population (29 in iduals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 in iduals. The remaining six in iduals were characterized by a high degree of sialylation. These in iduals were assigned as nonsecretors, whereas blood group-expressing in iduals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Le(a)) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between in iduals and that some of this variation is owing to blood group and secretor status.
Recombinant truncated AniA of pathogenic Neisseria elicits a non-native immune response and functional blocking antibodies
Publisher: Elsevier BV
Date: 02-2013
‘Omic’ Approaches to Study Uropathogenic Escherichia coli Virulence
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.TIM.2017.04.006
Abstract: Uropathogenic Escherichia coli (UPEC) is a pathogen of major significance to global human health and is strongly associated with rapidly increasing antibiotic resistance. UPEC is the primary cause of urinary tract infection (UTI), a disease that involves a complicated pathogenic pathway of extracellular and intracellular lifestyles during interaction with the host. The application of multiple 'omic' technologies, including genomics, transcriptomics, proteomics, and metabolomics, has provided enormous knowledge to our understanding of UPEC biology. Here we outline this progress and present a view for future developments using these exciting forefront technologies to fully comprehend UPEC pathogenesis in the context of infection.
Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae.
Publisher: American Society for Microbiology
Date: 15-05-2015
DOI: 10.1128/AEM.04144-14
Abstract: Monoterpenes are liquid hydrocarbons with applications ranging from flavor and fragrance to replacement jet fuel. Their toxicity, however, presents a major challenge for microbial synthesis. Here we evolved limonene-tolerant Saccharomyces cerevisiae strains and sequenced six strains across the 200-generation evolutionary time course. Mutations were found in the tricalbin proteins Tcb2p and Tcb3p. Genomic reconstruction in the parent strain showed that truncation of a single protein (tTcb3p 1-989 ), but not its complete deletion, was sufficient to recover the evolved phenotype improving limonene fitness 9-fold. tTcb3p 1-989 increased tolerance toward two other monoterpenes (β-pinene and myrcene) 11- and 8-fold, respectively, and tolerance toward the biojet fuel blend AMJ-700t (10% cymene, 50% limonene, 40% farnesene) 4-fold. tTcb3p 1-989 is the first ex le of successful engineering of phase tolerance and creates opportunities for production of the highly toxic C 10 alkenes in yeast.
A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure
Publisher: Springer Science and Business Media LLC
Date: 08-10-2014
DOI: 10.1007/S10529-013-1341-0
Abstract: The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.
Discovery and Characterisation of Castlerea Virus, a New Species of Negevirus Isolated in Australia
Publisher: SAGE Publications
Date: 2017
Abstract: With advances in sequencing technologies, there has been an increase in the discovery of viruses that do not group with any currently described virus families. The newly described taxon Negevirus encompasses a group of viruses displaying an insect-specific phenotype which have been isolated from multiple host species on numerous continents. Using a broad-spectrum virus screening assay based on the detection of double-stranded RNA and next-generation sequencing, we have detected a novel species of negevirus, from Anopheles, Culex, and Aedes mosquitoes collected in 4 geographically separate regions of Australia. Bioinformatic analysis of the virus, tentatively named Castlerea virus, revealed that it is genetically distinct from previously described negeviruses but clusters in the newly proposed Nelorpivirus clade within this taxon. Analysis of virions confirmed the presence of 2 proteins of 24 and 40 kDa which support previous bioinformatic predictions of negevirus structural proteins.
Proteomics and Metabolomics Reveal that an Abundant α-Glucosidase Drives Sorghum Fermentability for Beer Brewing
Publisher: American Chemical Society (ACS)
Date: 11-10-2023
Post-translational modifications drive protein stability to control the dynamic beer brewing proteome
Publisher: Cold Spring Harbor Laboratory
Date: 29-06-2018
DOI: 10.1101/358796
Abstract: Mashing is a key step in beer brewing in which starch and proteins are solubilized from malted barley in a hot water extraction and digested to oligomaltose and free amino nitrogen. We used SWATH-MS to measure the abundance and site-specific modifications of proteins throughout a small-scale pale ale mash. Proteins extracted from the malt at low temperatures early in the mash decreased precipitously in abundance at higher temperatures late in the mash due to temperature/time-induced unfolding and aggregation. We validated these observations using experimental manipulation of time and temperature parameters in a micro-scale pale ale mash. Correlation analysis of temperature/time-dependent abundance showed that sequence and structure were the main features that controlled protein abundance profiles. Partial proteolysis by barley proteases was common early in the mash. The resulting proteolytically clipped proteins were particularly sensitive and were preferentially lost at high temperatures late in the mash, while intact proteins remained soluble. The beer brewing proteome is therefore driven by the interplay between protein solubilisation and proteolysis, which are in turn determined by barley variety, growth conditions, and brewing process parameters.
Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears
Publisher: Portland Press Ltd.
Date: 09-2002
DOI: 10.1042/BJ20011876
Abstract: Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose olyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of & kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.
New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype
Publisher: Microbiology Society
Date: 04-2018
DOI: 10.1099/JGV.0.001038
Abstract: Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.
Sequential Analysis of N- and O-Linked Glycosylation of 2D-PAGE Separated Glycoproteins
Publisher: American Chemical Society (ACS)
Date: 07-09-2002
DOI: 10.1021/PR025538D
Abstract: A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha2-HS-glycoprotein, serotransferrin, alpha1-antitrypsin, and alpha1-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.
Acid Hydrolysis and Molecular Density of Phytoglycogen and Liver Glycogen Helps Understand the Bonding in Glycogen α (Composite) Particles
Publisher: Public Library of Science (PLoS)
Date: 23-03-2015
Epigenetic Regulation Alters Biofilm Architecture and Composition in Multiple Clinical Isolates of Nontypeable Haemophilus influenzae
Publisher: American Society for Microbiology
Date: 07-11-2018
Abstract: Upper respiratory tract infections are the number one reason for a child to visit the emergency department, and otitis media (middle ear infection) ranks third overall. Biofilms contribute significantly to the chronic nature of bacterial respiratory tract infections, including otitis media, and make these diseases particularly difficult to treat. Several mucosa-associated human pathogens utilize a mechanism of rapid adaptation termed the phasevarion, or phase vari able regul on , to resist environmental and host immune pressures. In this study, we assessed the role of the phasevarion in regulation of biofilm formation by nontypeable Haemophilus influenzae (NTHI), which causes numerous respiratory tract diseases. We found that the NTHI phasevarion regulates biofilm structure and critical biofilm matrix components under disease-specific conditions. The findings of this work could be significant in the design of improved strategies against NTHI infections, as well as diseases due to other pathogens that utilize a phasevarion.
High-Frequency Changes in Pilin Glycosylation Patterns during Neisseria meningitidis Serogroup a Meningitis Outbreaks in the African Meningitis Belt
Publisher: American Chemical Society (ACS)
Date: 19-07-2023
Identification of novel glycosylation events on human serum-derived Factor IX
Publisher: Cold Spring Harbor Laboratory
Date: 05-03-2019
DOI: 10.1101/567941
Abstract: Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O -linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc 1 Hex 1 NeuAc 2 dominant and a low level of HexNAc 1 Hex 1 NeuAc 1 . This O -linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N -linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc 4 Hex 5 NeuAc 2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.
Sequence-based protein stabilization in the absence of glycosylation
Publisher: Springer Science and Business Media LLC
Date: 17-01-2014
DOI: 10.1038/NCOMMS4099
Abstract: Asparagine-linked N-glycosylation is a common modification of proteins that promotes productive protein folding and increases protein stability. Although N-glycosylation is important for glycoprotein folding, the precise sites of glycosylation are often not conserved between protein homologues. Here we show that, in Saccharomyces cerevisiae, proteins upregulated during sporulation under nutrient deprivation have few N-glycosylation sequons and in their place tend to contain clusters of like-charged amino-acid residues. Incorporation of such sequences complements loss of in vivo protein function in the absence of glycosylation. Targeted point mutation to create such sequence stretches at glycosylation sequons in model glycoproteins increases in vitro protein stability and activity. A dependence on glycosylation for protein stability or activity can therefore be rescued with a small number of local point mutations, providing evolutionary flexibility in the precise location of N-glycans, allowing protein expression under nutrient-limiting conditions, and improving recombinant protein production.
Use of graphitised carbon negative ion LC–MS to analyse enzymatically digested glycosaminoglycans
Publisher: Elsevier BV
Date: 25-09-2005
DOI: 10.1016/J.JCHROMB.2005.07.014
Abstract: Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and s le preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid s les.
Quantitative Data-Independent Acquisition Glycoproteomics of Sparkling Wine
Publisher: Elsevier BV
Date: 2021
Circulating Fragments of N-Terminal Pro–B-Type Natriuretic Peptides in Plasma of Heart Failure Patients
Publisher: Oxford University Press (OUP)
Date: 10-2013
DOI: 10.1373/CLINCHEM.2012.200204
Abstract: The use of nonstandardized N-terminal pro–B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients. Plasma s les were collected from HF patients (n = 20) at rest and stored at −80 °C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA® immunoassays were developed and validated to quantify 6 fragments of NT-proBNP. Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P & 0.05). In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule.
Substrate recognition of a structure motif for phosphorylcholine post-translational modification in Neisseria meningitidis
Publisher: Elsevier BV
Date: 02-2013
DOI: 10.1016/J.BBRC.2012.12.088
Abstract: Neisseria meningitidis is a human pathogen that can cause life threatening meningitis and sepsis. Pili of Neisseria are one of the major virulence factors in host-pathogen interaction. Pilin of N.meningitidis is post-translationally modified by a glycan and two phosphorylcholines (ChoP). ChoP modifications have been found to have an important role in bacterial colonisation and invasion. Unlike N. gonorrhoeae, ChoP modifications on pili seem to be restricted to the C-terminus of pilin protein in N. meningitidis. In this study, we investigate the substrate recognition of phosphorylcholine transferase. We found that a single sequence of D-A-S after the disulphide bond of pilin protein is able to form a motif for ChoP modifications and the charge residue in this motif and the local structure are essential for the substrate recognition.
Molecular structure of glycogen in diabetic liver
Publisher: Springer Science and Business Media LLC
Date: 22-03-2015
DOI: 10.1007/S10719-015-9578-6
Abstract: Liver glycogen (involved in maintaining blood-sugar levels) is a hyperbranched glucose polymer containing β particles (diameter ~20 nm), which can form composite α particles (diameter ~50-300 nm), and includes a small but significant amount of bound protein. Size distributions of glycogen from livers of healthy and diabetic mice were examined using size-exclusion chromatography with two separate eluents: aqueous eluent and dimethylsulfoxide (DMSO) eluent. Morphologies were examined with transmission electron microscopy. Diabetic glycogen (DG) exhibited many α particles in the mild water-based solvent, but in DMSO, which breaks H bonds, these degraded to β particles α particles however were always present in healthy glycogen (HG). This DG fragility shows the binding of β into α particles is different in HG and DG. The diabetic α particle fragility may be involved with the uncontrolled blood-sugar release symptomatic of diabetes: small β particles degrade more easily to glucose than α particles. This has implications for diabetes management.
Analysis of mucosal mucins separated by SDS‐urea agarose polyacrylamide composite gel electrophoresis
Publisher: Wiley
Date: 24-11-2011
Abstract: Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose olyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin s les. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.
Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation
Publisher: Elsevier BV
Date: 12-2014
Dual Pili Post-translational Modifications Synergize to Mediate Meningococcal Adherence to Platelet Activating Factor Receptor on Human Airway Cells
Publisher: Public Library of Science (PLoS)
Date: 16-05-2013
The current status of heart failure diagnostic biomarkers
Publisher: Informa UK Limited
Date: 17-02-2016
DOI: 10.1586/14737159.2016.1144474
Abstract: Heart failure (HF) affects approximately 23 million in iduals worldwide and this number is increasing, due to an aging and growing population. Early detection of HF is crucial in the management of this debilitating disease. Current diagnostic methods for HF rely heavily on clinical imaging techniques and blood analysis, which makes them less than ideal for population-based screening purposes. Studies focusing on developing novel biomarkers for HF have utilized various techniques and biological fluids, including urine and saliva. Promising results from these studies imply that these body fluids can be used in evaluating the clinical manifestation of HF and will one day be integrated into a clinical workflow and facilitate HF management.
Globally elevating the AGE clearance receptor, OST48, does not protect against the development of diabetic kidney disease, despite improving insulin secretion
Publisher: Cold Spring Harbor Laboratory
Date: 22-07-2019
DOI: 10.1101/710558
Abstract: The accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic kidney disease (DKD). There has been interest in investigating the potential of AGE clearance receptors, such as oligosaccharyltransferase-48kDa subunit (OST48) to prevent the detrimental effects of excess AGE accumulation seen in the diabetic kidney. Here the objective of the study was to increase the expression of OST48 to examine if this slowed the development of DKD by facilitating the clearance of AGEs. Groups of 8-week-old heterozygous knock-in male mice (n=9-12/group) over-expressing the gene encoding for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase ( DDOST +/-) and litter mate controls were randomised to either (i) no diabetes or (ii) diabetes induced via multiple low-dose streptozotocin and followed for 24 weeks. By the study end, global over expression of OST48 increased glomerular OST48. This facilitated greater renal excretion of AGEs but did not affect circulating or renal AGE concentrations. Diabetes resulted in kidney damage including lower glomerular filtration rate, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. In diabetic mice, tubulointerstitial fibrosis was further exacerbated by global increases in OST48. There was significantly insulin effectiveness, increased acute insulin secretion, fasting insulin concentrations and AUC insulin observed during glucose tolerance testing in diabetic mice with global elevations in OST48 when compared to diabetic wild-type littermates. Overall, this study suggested that despite facilitating urinaryrenal AGE clearance, there were no benefits observed on kidney functional and structural parameters in diabetes afforded by globally increasing OST48 expression. However, the improvements in insulin secretion seen in diabetic mice with global over-expression of OST48 and their dissociation from effects on kidney function warrant future investigation.
The globally disseminated M1T1 clone of group a streptococcus evades autophagy for intracellular replication
Publisher: Elsevier BV
Date: 12-2013
Correction: Acid Hydrolysis and Molecular Density of Phytoglycogen and Liver Glycogen Helps Understand the Bonding in Glycogen α (Composite) Particles
Publisher: Public Library of Science (PLoS)
Date: 24-07-2015
A genomic safe haven for mutant complementation in cryptococcus neoformans
Publisher: Public Library of Science (PLoS)
Date: 09-04-2015
Vegemite Beer: yeast extract spreads as nutrient supplements to promote fermentation
Publisher: PeerJ
Date: 10-08-2016
DOI: 10.7717/PEERJ.2271
Abstract: Vegemite is an iconic Australian food spread made from spent brewers’ yeast extract, which has been reported to be used as an ingredient in illegal home brewing. In this study, we tested the utility of Vegemite and the similar spread Marmite in promoting fermentation. We could not culture microorganisms from either Vegemite or Marmite, consistent with these food-grade spreads being essentially sterile. To test if the addition of Vegemite or Marmite could assist in fermentation when additional viable yeast was also present, solutions containing glucose and a range of concentrations of either Vegemite or Marmite were inoculated with brewers’ yeast. No fermentation occurred in any condition without addition of extra brewer’s yeast. Fermentation did not occur when yeast was inoculated into solutions containing only glucose, but progressed efficiently with when Vegemite or Marmite was also added. Gas Chromatography confirmed that ethanol was present at ∼3% v/v post-fermentation in all s les which contained glucose, Vegemite or Marmite, and brewers’ yeast. Trace amounts of methanol were also detected. Mass spectrometry proteomics identified abundant intracellular yeast proteins and barley proteins in Vegemite and Marmite, and abundant secreted yeast proteins from actively growing yeast in those s les to which extra brewers’ yeast had been added. We estimate that the real-world cost of home brewed “Vegemite Beer” would be very low. Our results show that Vegemite or other yeast extract spreads could provide cheap and readily available sources of nutrient supplementation to increase the efficiency of fermentation in home brewing or other settings.
Increased liver AGEs induce hepatic injury mediated through an OST48 pathway
Publisher: Springer Science and Business Media LLC
Date: 25-09-2017
DOI: 10.1038/S41598-017-12548-4
Abstract: The protein oligosaccharyltransferase-48 (OST48) is integral to protein N-glycosylation in the endoplasmic reticulum (ER) but is also postulated to act as a membrane localised clearance receptor for advanced glycation end-products (AGE). Hepatic ER stress and AGE accumulation are each implicated in liver injury. Hence the objective of this study was to increase the expression of OST48 and examine the effects on hepatic function and structure. Groups of 8 week old male mice (n = 10–12/group) over-expressing the gene for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase ( DDOST +/−), were followed for 24 weeks, while randomised to diets either low or high in AGE content. By week 24 of the study, either increasing OST48 expression or consumption of high AGE diet impaired liver function and modestly increased hepatic fibrosis, but their combination significantly exacerbated liver injury in the absence of steatosis. DDOST +/− mice had increased both portal delivery and accumulation of hepatic AGEs leading to central adiposity, insulin secretory defects, shifted fuel usage to fatty and ketoacids, as well as hepatic glycogen accumulation causing hepatomegaly along with hepatic ER and oxidative stress. This study revealed a novel role of the OST48 and AGE axis in hepatic injury through ER stress, changes in fuel utilisation and glucose intolerance.
Identification of Bacterial Protein O-Oligosaccharyltransferases and Their Glycoprotein Substrates
Publisher: Public Library of Science (PLoS)
Date: 03-05-2013
Analysis of Congenital Disorder of Glycosylation-Id in a Yeast Model System Shows Diverse Site-Specific Under-glycosylation of Glycoproteins
Publisher: American Chemical Society (ACS)
Date: 16-10-2012
DOI: 10.1021/PR300599F
Abstract: Asparagine-linked glycosylation is a common post-translational modification of proteins in eukaryotes. Mutations in the human ALG3 gene cause changed levels and altered glycan structures on mature glycoproteins and are the cause of a severe congenital disorder of glycosylation (CDG-Id). Diverse glycoproteins are also under-glycosylated in Saccharomyces cerevisae alg3 mutants. Here we analyzed site-specific glycosylation occupancy in this yeast model system using peptide-N-glycosidase F to label glycosylation sites with an asparagine-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by LC-ESI-MS/MS. We used this analytical method to identify and measure site-specific glycosylation occupancy in alg3 mutant and wild type yeast strains. We found decreased site-specific N-glycosylation occupancy in the alg3 knockout strain preferentially at Asn-Xaa-Ser sequences located in secondary structural elements, features previously associated with poor glycosylation efficiency. Furthermore, we identified 26 previously experimentally unverified glycosylation sites. Our results provide insights into the underlying mechanisms of disease in CDG-Id, and our methodology will be useful in site-specific glycosylation analysis in many model systems and clinical applications.
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.
Fine-tuning the Spike: Role of the nature and topology of the glycan shield in the structure and dynamics of the SARS-CoV-2 S
Publisher: Cold Spring Harbor Laboratory
Date: 04-2021
DOI: 10.1101/2021.04.01.438036
Abstract: The dense glycan shield is an essential feature of the SARS-CoV-2 spike (S) architecture, key to immune evasion and to the activation of the prefusion conformation. Recent studies indicate that the occupancy and structures of the SARS-CoV-2 S glycans depend not only on the nature of the host cell, but also on the structural stability of the trimer a point that raises important questions about the relative competence of different glycoforms. Moreover, the functional role of the glycan shield in the SARS-CoV-2 pathogenesis suggests that the evolution of the sites of glycosylation is potentially intertwined with the evolution of the protein sequence to affect optimal activity. Our results from multi-microsecond molecular dynamics simulations indicate that the type of glycosylation at N234, N165 and N343 greatly affects the stability of the receptor binding domain (RBD) open conformation, and thus its exposure and accessibility. Furthermore, our results suggest that the loss of glycosylation at N370, a newly acquired modification in the SARS-CoV-2 S glycan shield’s topology, may have contributed to increase the SARS-CoV-2 infectivity as we find that N-glycosylation at N370 stabilizes the closed RBD conformation by binding a specific cleft on the RBD surface. We discuss how the absence of the N370 glycan in the SARS-CoV-2 S frees the RBD glycan binding cleft, which becomes available to bind cell-surface glycans, potentially increases host cell surface localization.
Metabolomics of Non-Saccharomyces Yeasts in Fermented Beverages
Publisher: MDPI AG
Date: 20-07-2022
Abstract: Fermented beverages have been consumed for millennia and today support a global industry producing erse products. Saccharomyces yeasts currently dominate the fermented beverage industry, but consumer demands for alternative products with a variety of sensory profiles and actual or perceived health benefits are driving the ersification and use of non-Saccharomyces yeasts. The ersity of flavours, aromas, and other sensory characteristics that can be obtained by using non-Saccharomyces yeasts in fermentation is, in large part, due to the erse secondary metabolites they produce compared to conventional Saccharomyces yeast. Here, we review the use of metabolomic analyses of non-Saccharomyces yeasts to explore their impact on the sensory characteristics of fermented beverages. We highlight several key species currently used in the industry, including Brettanomyces, Torulaspora, Lachancea, and Saccharomycodes, and emphasize the future potential for the use of non-Saccharomyces yeasts in the production of erse fermented beverages.
Applications of Ion Mobility-Mass Spectrometry in Carbohydrate Chemistry and Glycobiology
Publisher: MDPI AG
Date: 07-10-2018
DOI: 10.3390/MOLECULES23102557
Abstract: Carbohydrate analyses are often challenging due to the structural complexity of these molecules, as well as the lack of suitable analytical tools for distinguishing the vast number of possible isomers. The coupled technique, ion mobility-mass spectrometry (IM-MS), has been in use for two decades for the analysis of complex biomolecules, and in recent years it has emerged as a powerful technique for the analysis of carbohydrates. For carbohydrates, most studies have focused on the separation and characterization of isomers in biological s les. IM-MS is capable of separating isomeric ions by drift time, and further characterizing them by mass analysis. Applications of IM-MS in carbohydrate analysis are extremely useful and important for understanding many biological mechanisms and for the determination of disease states, although efforts are still needed for higher sensitivity and resolution.
Structural elements that modulate the substrate specificity of plant purple acid phosphatases: avenues for improved phosphorus acquisition in crops
Publisher: Cold Spring Harbor Laboratory
Date: 28-08-2019
DOI: 10.1101/749564
Abstract: Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. The crystal structure of this enzyme in complex with an ATP analogue (to 2.20 Å resolution) provides insight into the amino acid residues that play an essential role in binding this substrate. Homology modelling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and Arabidopsis thaliana (AtPAP12 and AtPAP26) that are efficient in hydrolyzing phytate and PEP as substrates, respectively. In combination with substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus ), several active site residues were identified that are important in defining the substrate specificities of plant PAPs. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production.
Recent advances in the production of recombinant factor IX: bioprocessing and cell engineering.
Publisher: Informa UK Limited
Date: 17-04-2022
DOI: 10.1080/07388551.2022.2036691
Abstract: Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells.
Quantitative data independent acquisition glycoproteomics of sparkling wine
Publisher: Cold Spring Harbor Laboratory
Date: 10-06-2020
DOI: 10.1101/2020.06.09.141226
Abstract: Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analysed the glyco roteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyse Sequential Window Acquisition of all THeoretical mass spectra (SWATH) Data Independent Acquisition (DIA) mass spectrometry data based on glycopeptides identified by Byonic. We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the Liqueur de Tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared to 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. This work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic SWATH/DIA workflow that is broadly applicable to other s le types.
The AGE receptor, OST48 drives podocyte foot process effacement and basement membrane expansion in experimental diabetic kidney disease via promotion of endoplasmic reticulum stress
Publisher: Cold Spring Harbor Laboratory
Date: 22-07-2019
DOI: 10.1101/710186
Abstract: The accumulation of advanced glycation end products is implicated in the development and progression of diabetic kidney disease. No study has examined whether stimulating advanced glycation clearance via receptor manipulation is reno-protective in diabetes. Podocytes, which are early contributors to diabetic kidney disease and could be a target for reno-protection. To examine the effects of increased podocyte oligosaccharyltransferase-48 on kidney function, glomerular sclerosis, tubulointerstitial fibrosis and proteome (PXD011434), we generated a mouse with increased oligosaccharyltransferase-48kDa subunit abundance in podocytes driven by the podocin promoter. Despite increased urinary clearance of advanced glycation end products, we observed a decline in renal function, significant glomerular damage including glomerulosclerosis, collagen IV deposition, glomerular basement membrane thickening and foot process effacement and tubulointerstitial fibrosis. Analysis of isolated glomeruli identified enrichment in proteins associated with collagen deposition, endoplasmic reticulum stress and oxidative stress. Ultra-resolution microscopy of podocytes revealed denudation of foot processes where there was co-localization of oligosaccharyltransferase-48kDa subunit and advanced glycation end-products. These studies indicate that increased podocyte expression of oligosaccharyltransferase-48kDa subunit results in glomerular endoplasmic reticulum stress and a decline in kidney function.
Mechanisms of Persistence of the Ammonia-Oxidizing Bacteria Nitrosomonas to the Biocide Free Nitrous Acid
Publisher: American Chemical Society (ACS)
Date: 05-04-2018
Abstract: Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA.
Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry
Publisher: Wiley
Date: 27-01-2014
DOI: 10.1002/RCM.6806
Abstract: Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively s led and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. Saliva was collected from healthy in iduals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy in iduals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. We developed a s le preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on erse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.
GlypNirO: An automated workflow for quantitative N- and O-linked glycoproteomic data analysis
Publisher: Cold Spring Harbor Laboratory
Date: 15-06-2020
DOI: 10.1101/2020.06.15.153528
Abstract: Mass spectrometry glycoproteomics is rapidly maturing, allowing unprecedented insights into the ersity and functions of protein glycosylation. However, quantitative glycoproteomics remains challenging. We developed GlypNirO, an automated software pipeline which integrates the complementary outputs of Byonic and Proteome Discoverer to allow high-throughput automated quantitative glycoproteomic data analysis. The output of GlypNirO is clearly structured, allowing manual interrogation, and is also appropriate for input into erse statistical workflows. We used GlypNirO to analyse a published plasma glycoproteome dataset and identified changes in site-specific N - and O -glycosylation occupancy and structure associated with hepatocellular carcinoma as putative biomarkers of disease.
Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn.
Publisher: American Chemical Society (ACS)
Date: 05-2004
S-Trap eliminates cell culture media polymeric surfactants for effective proteomic analysis of mammalian cell bioreactor supernatants
Publisher: Cold Spring Harbor Laboratory
Date: 18-02-2020
DOI: 10.1101/2020.02.17.951798
Abstract: Proteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during s le preparation. Here, we compared four different s le preparation methods to eliminate polymeric surfactants from filtered bioreactor supernatant s les: organic solvent precipitation filter-assisted s le preparation (FASP) S-Trap and single-pot, solid-phase, s le preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust clean-up and highest quality data. Additionally, we observed that SP3 s le preparation of our s les and in other published datasets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with similar mass spectrometry profiles, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap s le preparation.
Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product
Publisher: Springer Science and Business Media LLC
Date: 23-03-2021
DOI: 10.1038/S42003-021-01903-X
Abstract: Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.
Structure and antigenicity of divergent Henipavirus fusion glycoproteins
Publisher: Springer Science and Business Media LLC
Date: 16-06-2023
DOI: 10.1038/S41467-023-39278-8
Abstract: In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiāng virus (MojV), and both are ergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence ergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, ergence from prototypical HNVs.
The post-translational modification landscape of commercial beers
Publisher: Cold Spring Harbor Laboratory
Date: 28-01-2021
DOI: 10.1101/2021.01.27.427706
Abstract: Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and ersity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high ersity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O -glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco roteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco roteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco roteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.
Benchtop micro-mashing: high-throughput, robust, experimental beer brewing
Publisher: Cold Spring Harbor Laboratory
Date: 24-06-2020
DOI: 10.1101/2020.06.24.158345
Abstract: Brewing science is undergoing a renaissance with the use of modern analytical chemistry and microbiology techniques. However, these modern analytical tools and techniques are not necessarily aligned with the scale and scope of brewing science. In particular, brewing processes can be time consuming, ingredient intensive, and require specialised technical equipment. These drawbacks compound with the need for appropriate numbers of replicates for adequately powered experimental design. Here, we describe a micro-scale mash method that can be performed using a common laboratory benchtop shaker/incubator, allowing for high throughput mashing and easy s le replication for statistical analysis. Proteomic profiles at both the protein and peptide levels were consistent between the 1 mL micro-mash and a 23 L Braumeister mash, and both mash scales produced wort with equivalent fermentable sugar and free amino acid profiles. The experimental flexibility offered by our micro-mash method allowed us to investigate the effects of altered mash parameters on the beer brewing proteome.
Adipose tissue proteomic analyses to study puberty in Brahman heifers
Publisher: Oxford University Press (OUP)
Date: 17-05-2018
DOI: 10.1093/JAS/SKY128
Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p
Publisher: Wiley
Date: 04-04-2011
DOI: 10.1002/PRO.610
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
Glycopeptide variable window SWATH for improved Data Independent Acquisition glycoprotein analysis
Publisher: Cold Spring Harbor Laboratory
Date: 19-08-2019
DOI: 10.1101/739615
Abstract: N -glycosylation plays an essential role in regulating protein folding and function in eukaryotic cells. Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH) has proven useful as a data independent acquisition (DIA) MS method for analysis of glycoproteins and their glycan modifications. By separating the entire m/z range into consecutive isolation windows, DIA-MS allows comprehensive MS data acquisition and high-sensitivity detection of molecules of interest. Variable width DIA windows allow optimal analyte measurement, as peptide ions are not evenly distributed across the full m/z range. However, the m/z distribution of glycopeptides is different to that of unmodified peptides because of their large glycan structures. Here, we improved the performance of DIA glycoproteomics by using variable width windows optimized for glycopeptides. This method allocates narrow windows at m/z ranges rich in glycopeptides, improving analytical specificity and performance. We show that related glycoforms must fall in separate windows to allow accurate glycopeptide measurement. We demonstrate the utility of the method by comparing the cell wall glycoproteomes of wild-type and N -glycan biosynthesis deficient yeast and showing improved measurement of glycopeptides with different glycan structures. Our results highlight the importance of appropriately optimized DIA methods for measurement of post-translationally modified peptides.
Identification of novel glycosylation events on human serum-derived factor IX
Publisher: Springer Science and Business Media LLC
Date: 06-05-2020
Selection against glycosylation sites in potential target proteins of the general HMWC N-glycosyltransferase in Haemophilus influenzae
Publisher: Elsevier BV
Date: 03-2014
DOI: 10.1016/J.BBRC.2014.02.044
Abstract: The HMWABC system of non-typeable Haemophilus influenzae (NTHi) encodes the HMWA adhesin glycoprotein, which is glycosylated by the HMWC glycosyltransferase. HMWC is a cytoplasmic N-glycosyltransferase, homologues of which are widespread in the Pasteurellaceae. We developed an assay for nonbiased detection of glycoproteins in NTHi based on metabolic engineering of the Leloir pathway and growth in media containing radiolabelled monosaccharides. The only glycoprotein identified in NTHi by this assay was HMWA. However, glycoproteomic analyses ex vivo in Escherichia coli showed that HMWC of NTHi was a general glycosyltransferase capable of glycosylating selected asparagines in proteins other than its HMWA substrate, including Asn78 in E. coli 30S ribosomal protein S5. The equivalent residue in S5 homologues in H. influenzae or other sequenced Pasteurellaceae genomes is not asparagine, and these organisms also showed significantly fewer than expected potential sites of glycosylation in general. Expression of active HMWC in E. coli resulted in growth inhibition compared with expression of inactive enzyme, consistent with glycosylation by HMWC detrimentally affecting the function of some E. coli proteins. Together, this supports the presence of a selective pressure in the Pasteurellaceae against glycosylation sites that would be modified by the general N-glycosyltransferase activity of HMWC.
High-Antibody-Producing Chinese Hamster Ovary Cells Up-Regulate Intracellular Protein Transport and Glutathione Synthesis
Publisher: American Chemical Society (ACS)
Date: 14-01-2015
DOI: 10.1021/PR501027C
Abstract: Chinese hamster ovary (CHO) cells are the preferred production host for therapeutic monoclonal antibodies (mAb) due to their ability to perform post-translational modifications and their successful approval history. The completion of the genome sequence for CHO cells has reignited interest in using quantitative proteomics to identify markers of good production lines. Here we applied two different proteomic techniques, iTRAQ and SWATH, for the identification of expression differences between a high- and low-antibody-producing CHO cell lines derived from the same transfection. More than 2000 proteins were quantified with 70 of them classified as differentially expressed in both techniques. Two biological processes were identified as differentially regulated by both methods: up-regulation of glutathione biosynthesis and down-regulation of DNA replication. Metabolomic analysis confirmed that the high producing cell line displayed higher intracellular levels of glutathione. SWATH further identified up-regulation of actin filament processes and intracellular transport and down regulation of several growth-related processes. These processes may be important for conferring high mAb production and as such are promising candidates for targeted engineering of high-expression cell lines.
CRISPR-knockout of β-kafirin in sorghum does not recapitulate the grain quality of natural mutants
Publisher: Springer Science and Business Media LLC
Date: 08-12-2022
Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils
Publisher: American Chemical Society (ACS)
Date: 21-07-2016
Signal sequence non-optimal codons are required for the correct folding of mature maltose binding protein
Publisher: Elsevier BV
Date: 06-2010
DOI: 10.1016/J.BBAMEM.2010.03.010
Abstract: Non-optimal codons are generally characterised by a low concentration of isoaccepting tRNA and a slower translation rate compared to optimal codons. In a previous study, we reported a 20-fold reduction in maltose binding protein (MBP) level when the non-optimal codons in the signal sequence were optimised. In this study, we report that the 20-fold reduction is rescued when MBP is expressed at 28 degrees C instead of 37 degrees C, suggesting that the signal sequence optimised MBP protein (MBP-opt) may be misfolded, and is being degraded at 37 degrees C. Consistent with this idea, transient induction of the heat shock proteases prior to MBP expression at 28 degrees C restores the 20-fold difference, demonstrating that the difference in production levels is due to post-translational degradation of MBP-opt by the heat-shock proteases. Analysis of the structure of purified MBP-wt and MBP-opt grown at 28 degrees C showed that although they have similar secondary structure content, MBP-opt is more resistant to thermal unfolding than is MBP-wt. The two proteins also exhibit different tryptic fragment profiles, further confirming that they are folded into conformationally different states. This is the first study to demonstrate that signal sequence non-optimal codons can influence the folding of the mature exported protein.
Process proteomics of beer reveals a dynamic proteome with extensive modifications
Publisher: Cold Spring Harbor Laboratory
Date: 20-12-2017
DOI: 10.1101/234252
Abstract: Modern beer production is a complex industrial process. However, some of its biochemical details remain unclear. Using mass spectrometry proteomics, we have performed a global untargeted analysis of the proteins present across time during nano-scale beer production. S les included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. This analysis identified over 200 unique proteins from barley and yeast, emphasizing the complexity of the process and product. We then used data independent SWATH-MS to quantitatively compare the relative abundance of these proteins throughout the process. This identified large and significant changes in the proteome at each process step. These changes described enrichment of proteins by their biophysical properties, and identified the appearance of dominant yeast proteins during fermentation. Altered levels of malt modification also quantitatively changed the proteomes throughout the process. Detailed inspection of the proteomic data revealed that many proteins were modified by protease digestion, glycation, or oxidation during the processing steps. This work demonstrates the opportunities offered by modern mass spectrometry proteomics in understanding the ancient process of beer production.
A rapid extraction method for glycogen from formalin-fixed liver.
Publisher: Elsevier BV
Date: 03-2015
DOI: 10.1016/J.CARBPOL.2014.11.005
Abstract: Liver glycogen, a highly branched polymer, acts as our blood-glucose buffer. While past structural studies have extracted glycogen from fresh or frozen tissue using a cold-water, sucrose-gradient centrifugation technique, a method for the extraction of glycogen from formalin-fixed liver would allow the analysis of glycogen from human tissues that are routinely collected in pathology laboratories. In this study, both sucrose-gradient and formalin-fixed extraction techniques were carried out on piglet livers, with the yields, purities and size distributions (using size exclusion chromatography) compared. The formalin extraction technique, when combined with a protease treatment, resulted in higher yields (but lower purities) of glycogen with size distributions similar to the sucrose-gradient centrifugation technique. This formalin extraction procedure was also significantly faster, allowing glycogen extraction throughput to increase by an order of magnitude. Both extraction techniques were compatible with mass spectrometry proteomics, with analysis showing the two techniques were highly complementary.
DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides
Publisher: Cold Spring Harbor Laboratory
Date: 10-07-2019
DOI: 10.1101/696518
Abstract: Data Independent Acquisition (DIA) Mass Spectrometry (MS) workflows allow unbiased measurement of all detectable peptides from complex proteomes, but require ion libraries for interrogation of peptides of interest. These DIA ion libraries can be theoretical or built from peptide identification data from Data Dependent Acquisition (DDA) MS workflows. However, DDA libraries derived from empirical data rely on confident peptide identification, which can be challenging for peptides carrying complex post-translational modifications. Here, we present DIALib, software to automate the construction of peptide and glycopeptide Data Independent Acquisition ion Libraries. We show that DIALib theoretical ion libraries can identify and measure erse N - and O -glycopeptides from yeast and mammalian glycoproteins without prior knowledge of the glycan structures present. We present proof-of-principle data from a moderately complex yeast cell wall glycoproteome and a simple mixture of mammalian glycoproteins. We also show that DIALib libraries consisting only of glycan oxonium ions can quickly and easily provide a global compositional glycosylation profile of the detectable “oxoniome” of glycoproteomes. DIALib will help enable DIA glycoproteomics as a complementary analytical approach to DDA glycoproteomics.
Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions
Publisher: Portland Press Ltd.
Date: 26-04-2005
DOI: 10.1042/BJ20041641
Abstract: SMG (submucosal gland) secretions are a major component of the airway surface liquid, are associated with innate immunity in the lung, and have been reported to be altered in lung disease. Changes in lung mucosal glycosylation have been reported in CF (cystic fibrosis), which may be responsible for differential bacterial binding to glycosylated components in the lung mucosa and hence increased pre-disposition to pulmonary infection. Glycoproteomic analysis was performed on SMG secretions collected from explanted bronchial tissue of subjects with severe lung disease, with and without CF, and controls without lung disease. Mucins MUC5B and MUC5AC were shown to be the dominant high-molecular-mass glycoprotein components, with a minor non-mucin glycoprotein component, gp-340, also present. Oligosaccharides containing blood-group determinants corresponding to subjects' blood type were abundant on MUC5B/MUC5AC, as were Lewis-type epitopes and their sialylated analogues, which are ligands for pathogens and leucocytes. No significant differences were found in the glycosylation of MUC5B/MUC5AC or gp-340 between CF and non-CF subjects with severe lung disease, implying that CF does not influence SMG secretion mucin glycosylation in end-stage lung disease. There were also no significant differences found in the glycosylation of these components in severe lung disease compared with non-diseased lungs. This suggests that previously reported changes in the glycosylation of respiratory glycoconjugates in CF, and other pulmonary conditions, are not due to the glycosylation of components in SMG secretions, but may involve other secretions, responses or extracellular factors.
Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product
Publisher: Cold Spring Harbor Laboratory
Date: 03-06-2020
DOI: 10.1101/2020.06.03.131177
Abstract: Coagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX post-translational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.
Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p
Publisher: Elsevier BV
Date: 03-2013
DOI: 10.1016/J.BBRC.2013.01.128
Abstract: Oligosaccharyltransferase (OTase) glycosylates selected asparagine residues in secreted and membrane proteins in eukaryotes, and asparagine (N)-glycosylation affects the folding, stability and function of erse glycoproteins. The range of acceptor protein substrates that are efficiently glycosylated depends on the action of several accessory subunits of OTase, including in yeast the homologous proteins Ost3p and Ost6p. A model of Ost3p and Ost6p function has been proposed in which their thioredoxin-like active site cysteines form transient mixed disulfide bonds with cysteines in substrate proteins to enhance the glycosylation of nearby asparagine residues. We tested aspects of this model with a series of in vitro assays. We developed a whole protein mixed disulfide interaction assay that showed that Ost6p could form mixed disulfide bonds with selected cysteines in pre-reduced yeast Gas1p, a model glycoprotein substrate of Ost3p and Ost6p. A complementary peptide affinity chromatography assay for mixed disulfide bond formation showed that Ost3p could also form mixed disulfide bonds with cysteines in selected reduced tryptic peptides from Gas1p. Together, these assays showed that the thioredoxin-like active sites of Ost3p and Ost6p could form transient mixed disulfide bonds with cysteines in a model substrate glycoprotein, consistent with the function of Ost3p and Ost6p in modulating N-glycosylation substrate selection by OTase in vivo.
A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction
Publisher: American Society for Microbiology
Date: 30-08-2017
Abstract: Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes - and Culex -associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.
Deciphering the electric code of Geobacter sulfurreducens in cocultures with Pseudomonas aeruginosa via SWATH-MS proteomics
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.BIOELECHEM.2017.09.013
Abstract: Interspecies electron transfer (IET) occurs in many microbial communities, enabling extracellular electron exchange for syntrophic utilization of mixed resources. Various mechanisms of IET have been characterized including direct IET (DIET) and hydrogen IET (HIT) but their evolution throughout syntrophic adaptation has not been investigated through an omics approach. A syntrophic coculture of Geobacter sulfurreducens and Pseudomonas aeruginosa was established and evolved in restricted medium. The medium required cooperative metabolism due to preferential utilization of formate and fumarate by P. aeruginosa and G. sulfurreducens respectively. Pure cultures did not yield significant growth while substantial growth was observed in cocultures. The syntrophy was not reliant on phenazine, since Δphz mutant strain cocultures grew, however appeared to rely on cytochromes as evidenced from the stunted growth G. sulfurreducens ΔomcZ and ΔomcS mutant cocultures. SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) proteomic analysis of initial cocultures revealed upregulation in DIET-associated cytochromes, whereas adapted cocultures revealed upregulation in HybA, a G. sulfurreducens uptake hydrogenase critical to HIT. This suggests DIET plays a critical role in the establishment of syntrophy between G. sulfurreducens and P. aeruginosa but is later consolidated with HIT as the cocultures adapt. This is the first instance to show a temporal distribution of DIET and HIT within the same coculture.
Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease
Publisher: Elsevier BV
Date: 06-2016
The role ofN-glycosylation in spike antigenicity for the SARS-CoV-2 Gamma variant
Publisher: Cold Spring Harbor Laboratory
Date: 07-04-2023
DOI: 10.1101/2023.04.03.535004
Abstract: The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impact N -glycosylation of spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additional N -glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody binding. Glycan site-occupancy analyses revealed that the mutation resulted in N -glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral ergence on spike glycosylation and SARS-CoV-2 antibody binding profiles.
N-glycosylation on Oryza Sativa Root Germin-like Protein 1 is conserved but not required for stability or activity
Publisher: Cold Spring Harbor Laboratory
Date: 10-02-2021
DOI: 10.1101/2021.02.09.430526
Abstract: Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 ( Os RGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of Os RGLP1 to study the role of glycosylation on Os RGLP1 protein stability and activity. Sequence analysis of Os RGLP1 homologs identified erse N -glycosylation sequons, one of which was highly conserved. We therefore expressed Os RGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified Os RGLP1 showed it was expressed by S. cerevisiae in both N -glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP- Os RGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP- Os RGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for Os RGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that Os RGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251±70 Units/mg. In conclusion, we find that some highly conserved N -glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of Os RGLP1 which paves the way for further characterization and use of this protein.
Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation
Publisher: The Company of Biologists
Date: 2020
DOI: 10.1242/JCS.243790
Abstract: The Endoplasmic Reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosacharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalyzed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. Thereby, RHBDL4 controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.
Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.JCHROMB.2012.10.023
Abstract: Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high ersity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid s le preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme ersity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva.
Proteomics Reveals Profound Metabolic Changes in the Alcohol Use Disorder Brain
Publisher: American Chemical Society (ACS)
Date: 26-02-2019
DOI: 10.1021/ACSCHEMNEURO.8B00660
Abstract: Changes in brain metabolism are a hallmark of alcohol use disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in prefrontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.
S-Trap Eliminates Cell Culture Media Polymeric Surfactants for Effective Proteomic Analysis of Mammalian Cell Bioreactor Supernatants
Publisher: American Chemical Society (ACS)
Date: 24-03-2020
Human blood microRNA hsa-miR-21-5p induces vitellogenin in the mosquito Aedes aegypti
Publisher: Springer Science and Business Media LLC
Date: 09-07-2202
DOI: 10.1038/S42003-021-02385-7
Abstract: Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti , a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another ex le of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa.
GlypNirO: An automated workflow for quantitative N- and O-linked glycoproteomic data analysis
Publisher: Beilstein Institut
Date: 09-2020
DOI: 10.3762/BJOC.16.180
Abstract: Mass spectrometry glycoproteomics is rapidly maturing, allowing unprecedented insights into the ersity and functions of protein glycosylation. However, quantitative glycoproteomics remains challenging. We developed GlypNirO, an automated software pipeline which integrates the complementary outputs of Byonic and Proteome Discoverer to allow high-throughput automated quantitative glycoproteomic data analysis. The output of GlypNirO is clearly structured, allowing manual interrogation, and is also appropriate for input into erse statistical workflows. We used GlypNirO to analyse a published plasma glycoproteome dataset and identified changes in site-specific N - and O -glycosylation occupancy and structure associated with hepatocellular carcinoma as putative biomarkers of disease.
Proteomics reveals profound metabolic changes in the alcohol use disorder brain
Publisher: Cold Spring Harbor Laboratory
Date: 20-10-2018
DOI: 10.1101/447912
Abstract: Changes in brain metabolism are a hallmark of Alcohol Use Disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in pre-frontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.
Glycosylation and the global virome.
Publisher: Wiley
Date: 21-10-2022
DOI: 10.1111/MEC.16731
Abstract: The sugars that coat the outsides of viruses and host cells are key to successful disease transmission, but they remain understudied compared to other molecular features. Understanding the comparative zoology of glycosylation - and harnessing it for predictive science - could help close the molecular gap in zoonotic risk assessment.
Structure aided design of a Neu5Gc specific lectin.
Publisher: Springer Science and Business Media LLC
Date: 04-05-2017
DOI: 10.1038/S41598-017-01522-9
Abstract: Subtilase cytotoxin (SubAB) of Escherichia coli is an AB5 class bacterial toxin. The pentameric B subunit (SubB) binds the cellular carbohydrate receptor, α2–3-linked N -glycolylneuraminic acid (Neu5Gc). Neu5Gc is not expressed on normal human cells, but is expressed by cancer cells. Elevated Neu5Gc has been observed in breast, ovarian, prostate, colon and lung cancer. The presence of Neu5Gc is prognostically important, and correlates with invasiveness, metastasis and tumour grade. Neu5Gc binding by SubB suggests that it may have utility as a diagnostic tool for the detection Neu5Gc tumor antigens. Native SubB has 20-fold less binding to N-acetlylneuraminic acid (Neu5Ac) over 30-fold less if the Neu5Gc linkage was changed from α2–3 to α2–6. Using molecular modeling approaches, site directed mutations were made to reduce the α2–3 $${\\boldsymbol{\\gg }}$$ ≫ α2–6-linkage preference, while maintaining or enhancing the selectivity of SubB for Neu5Gc over Neu5Ac. Surface plasmon resonance and glycan array analysis showed that the SubBΔS106/ΔT107 mutant displayed improved specificity towards Neu5Gc and bound to α2–6-linked Neu5Gc. SubBΔS106/ΔT107 could discriminate NeuGc- over Neu5Ac-glycoconjugates in ELISA. These data suggest that improved SubB mutants offer a new tool for the testing of biological s les, particularly serum and other fluids from in iduals with cancer or suspected of having cancer.
Diminished Ost3-dependent N-glycosylation of the BiP nucleotide exchange factor Sil1 is an adaptive response to reductive ER stress
Publisher: Proceedings of the National Academy of Sciences
Date: 06-11-2017
Abstract: Endoplasmic reticulum (ER) function relies on the maintenance of an appropriate redox balance. We observe that ER reductive stress is sensed through the oligosaccharyltransferase subunit Ost3 and acts to inhibit glycosylation of Sil1. Misfolded proteins accumulate within the ER under reductive stress and may sequester Lhs1 and limit the pool of free Lhs1 that can function as a BiP nucleotide exchange factor. Furthermore, unglycosylated Sil1 can compensate for the loss of Lhs1 activity more readily than glycosylated Sil1. Accumulation of unglycosylated Sil1 upon reductive stress may therefore be an adaptive mechanism that allows Sil1 to compensate for loss of Lhs1 NEF activity under these conditions.
Glycosylation of sputum mucins is altered in cystic fibrosis patients
Publisher: Oxford University Press (OUP)
Date: 28-03-2007
Abstract: Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after s ling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.
DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/C9MO00125E
Abstract: Data Independent Acquisition (DIA) Mass Spectrometry (MS) workflows allow unbiased measurement of all detectable peptides from complex proteomes, but require ion libraries for interrogation of peptides of interest.
Proteomics and glycoproteomics of beer and wine
Publisher: Wiley
Date: 26-06-2022
Abstract: Beer and wine are fermented beverages that contain abundant proteins released from barley or grapes, and secreted from yeast. These proteins are associated with many quality attributes including turbidity, foamability, effervescence, flavour and colour. Many grape proteins and secreted yeast proteins are glycosylated, and barley proteins can be glycated under the high temperatures in the beer making process. The emergence of high‐resolution mass spectrometry has allowed proteomic and glycoproteomic analyses of these complex mixtures of proteins towards understanding their role in determining beer and wine attributes. In this review, we summarise recent studies of proteomic and glycoproteomic analyses of beer and wine including their strategies for mass spectrometry (MS)‐based identification, quantification and characterisation of the glyco roteomes of fermented beverages to control product quality.
Proteomics reveals commitment to germination in barley seeds is marked by loss of stress response proteins and mobilisation of nutrient reservoirs
Publisher: Cold Spring Harbor Laboratory
Date: 02-06-2020
DOI: 10.1101/2020.06.02.130823
Abstract: Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germination of the seeds before harvest from the field. This can result in inefficiencies in industrial malting. We used Sequential Window Acquisition of all THeoretical ions Mass Spectrometry (SWATH-MS) proteomics to measure changes in the barley seed proteome throughout germination. We found a large number of proteins involved in desiccation tolerance and germination inhibition rapidly decreased in abundance after imbibition. This was followed by a decrease in proteins involved in lipid, protein and nutrient reservoir storage, consistent with induction and activation of systems for nutrient mobilisation to provide nutrients to the growing embryo. Dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, with differences in sulfur metabolic enzymes, endogenous alpha-amylase/trypsin inhibitors, and histone proteins. We verified our findings with analysis of germinating barley seeds from two commercial malting facilities, demonstrating that key features of the dynamic proteome of germinating barley seeds were conserved between laboratory and industrial scales. The results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy.
High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity
Publisher: Elsevier BV
Date: 10-2016
Abstract: Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM(HR)) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest s le. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.
A Unique Relative of Rotifer Birnavirus Isolated from Australian Mosquitoes
Publisher: MDPI AG
Date: 22-09-2020
DOI: 10.3390/V12091056
Abstract: The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.
A new non-degradative method to purify glycogen
Publisher: Elsevier BV
Date: 08-2016
DOI: 10.1016/J.CARBPOL.2016.04.009
Abstract: Liver glycogen, a complex branched glucose polymer containing a small amount of protein, is important for maintaining glucose homeostasis (blood-sugar control) in humans. It has recently been found that glycogen molecular structure is impaired in diabetes. Isolating the carbohydrate polymer and any intrinsically-attached protein(s) is an essential prerequisite for studying this structural impairment. This requires an effective, non-degradative and efficient purification method to exclude the many other proteins present in liver. Proteins and glycogen have different ranges of molecular sizes. Despite the plethora of proteins that might still be present in significant abundance after other isolation techniques, SEC (size exclusion chromatography, also known as GPC), which separates by molecular size, should separate those extraneous to glycogen from glycogen with any intrinsically associated protein(s). A novel purification method is developed for this, based on preparative SEC following sucrose gradient centrifugation. Proteomics is used to show that the new method compares favourably with current methods in the literature.
Cell wall and whole cell proteomes define flocculation and fermentation behavior of yeast
Publisher: MDPI AG
Date: 19-09-2018
DOI: 10.3390/FERMENTATION4030080
Abstract: Flocculation is one of the most important characteristics of brewing yeast as it allows for the easy and cheap removal of cells after fermentation. The genes responsible for both the Flo1 and NewFlo flocculation phenotypes are well characterized. However, the relationship between Flo protein abundance and flocculation efficiency is poorly understood. In this present study, we used mass spectrometry proteomics to compare the cell wall and whole cell proteomes of commercial yeast strains with erse flocculation behaviors. We found that the relative abundance of Flo1/5 or Flo10 in the cell wall was correlated with the ability of these yeast strains to flocculate. Analysis of whole cell proteomes identified differences in the proteomes of yeast strains and identified the potential for high metabolic ersity. Characterization of the cell wall and whole cell proteomes during fermentation showed high levels of Flo10 in cells that settled early during fermentation. Our data reveal the ersity of the cell wall and global proteomes of brewing yeast, highlighting the potential biochemical ersity present in yeast that can be utilized in the production of fermented beverages.
Small-Scale Analysis of O-Linked Oligosaccharides from Glycoproteins and Mucins Separated by Gel Electrophoresis
Publisher: American Chemical Society (ACS)
Date: 17-10-2002
DOI: 10.1021/AC025890A
Abstract: A technique with subpicomolar sensitivity was developed for analyzing O-linked oligosaccharides released from glycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly-(vinylidene fluoride) membrane, reductive beta-elimination, and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray mass spectrometry. It was also found that N-linked oligosaccharides could be recovered under the same conditions, found both as free oligosaccharides and as distinct glycopeptides created from reductive cleavage of the protein backbone, giving some information on site-specific glycosylation. The method was used to demonstrate that the difference between human alpha-2HS-glycoprotein isoforms separated by 2D-gel electrophoresis was partially due to sialylation of both O-linked and N-linked oligosaccharides. It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecular mass (200,000-5,000,000 Da) highly glycosylated mucin glycoproteins collected from small intestine and saliva and separated by sodium dodecyl sulfate-agarose olyacrylamide composite gels. Mass spectrometric data not only gave information about the mass distribution of the heterogeneous mixtures of oligosaccharides from [M - xH](x-) ions but also gave information about the isomeric heterogeneity of the oligosaccharides from their resolution by porous graphitized carbon chromatography. Tandem mass spectrometry was explored as a technique for distinguishing between oligosaccharide isomers with different sequences and also between oligosaccharides with the same sequence but with different linkage configurations.
Development of a mass fingerprinting tool for automated interpretation of oligosaccharide fragmentation data
Publisher: Wiley
Date: 25-05-2004
Abstract: The bioinformatic tool GlycosidIQ was developed for computerized interpretation of oligosaccharide mass spectrometric fragmentation based on matching experimental data with theoretically fragmented oligosaccharides generated from the database GlycoSuiteDB. This use of the software for glycofragment mass fingerprinting obviates a large part of the manual, labor intensive, and technically challenging interpretation of oligosaccharide fragmentation. Using 130 negative ion electrospray ionization-tandem mass spectrometry fragment spectra from identified oligosaccharide structures, it was shown that the GlycosidIQ scoring algorithms were able to correctly identify oligosaccharides in the great majority of cases (correct structure top ranked in 78% of the cases and an additional 17% were ranked second highest in the s le set).
Pre‐ and post‐puberty expression of genes and proteins in the uterus of Bos indicus heifers: the luteal phase effect post‐puberty
Publisher: Wiley
Date: 07-09-2018
DOI: 10.1111/AGE.12721
Abstract: Progesterone signaling and uterine function are crucial in terms of pregnancy establishment. To investigate how the uterine tissue and its secretion changes in relation to puberty, we s led tissue and uterine fluid from six pre- and six post-pubertal Brahman heifers. Post-pubertal heifers were s led in the luteal phase. Gene expression of the uterine tissue was investigated with RNA-sequencing, whereas the uterine fluid was used for protein profiling with mass spectrometry. A total of 4034 genes were differentially expressed (DE) at a nominal P-value of 0.05, and 26 genes were significantly DE after Bonferroni correction (P < 3.1 × 10
Identification and Validation of a Salivary Protein Panel to Detect Heart Failure Early
Publisher: Ivyspring International Publisher
Date: 2017
DOI: 10.7150/THNO.21727
Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases
Publisher: Wiley
Date: 23-07-2009
Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays
Publisher: Elsevier BV
Date: 03-2016
Abstract: Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the s le collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical s les is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.
Proteomic Investigation of the Binding Agent between Liver Glycogen β Particles
Publisher: American Chemical Society (ACS)
Date: 02-04-2018
Proteomics and metabolomics reveal an abundant α-glucosidase drives sorghum fermentability for beer brewing
Publisher: Cold Spring Harbor Laboratory
Date: 07-2022
DOI: 10.1101/2022.06.30.498347
Abstract: Sorghum ( Sorghum bicolor ), a grass native to Africa, is a popular alternative to barley for brewing beer. The importance of sorghum to beer brewing is increasing because it is a naturally gluten-free cereal and because climate change is expected to cause a reduction in the production of barley over the coming decades. However, there are challenges associated with the use of sorghum instead of barley in beer brewing. Here, we used proteomics and metabolomics to gain insights into the sorghum brewing process, to advise processes for efficient beer production from sorghum. We found that during malting, sorghum synthesises the amylases and proteases necessary for brewing. Proteomics revealed that mashing with sorghum malt required higher temperatures than barley malt for efficient protein solubilisation. Both α- and β-amylase were considerably less abundant in sorghum wort than in barley wort, correlating with lower maltose concentrations in sorghum wort. However, metabolomics revealed higher glucose concentrations in sorghum wort than in barley wort, consistent with the presence of an abundant α-glucosidase detected by proteomics in sorghum malt. Our results indicate that sorghum can be a viable grain for industrial fermented beverage production, but that its use requires careful process optimisation for efficient production of fermentable wort and high-quality beer.
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