Biology subjects

Ashwood, C.

Publications and source records attributed to Ashwood, C..

11 recordsLinked to original sources

bioRxiv2026

Development of a Matrix-Matched Calibration Curve for Multi-Site Quantification of Neu5Gc-Bearing N-Glycans

N-glycolylneuraminic acid (Neu5Gc) has been repeatedly associated with human cancer, but reliable detection has remained elusive, generating controversy regarding its presence in human samples. To address this, matrix-matched calibration curves, which have been pioneered in proteomics and metabolomics for assessing changes in complex mixtures, were measured of released N-glycans at four orders of magnitude dynamic range in defined mixtures, systematically benchmarking Neu5Gc-containing N-glycan detection across multiple LC-MS platforms and sites. Orthogonally, the gold-standard analytical method, consisting of fluorescence detection of labelled monosaccharides separated by LC, was applied to the same samples, yielding absolute concentrations of Neu5Gc. LC-MS demonstrated an extended detection range of three or more orders of magnitude while retaining intact N-glycan measurement, improving assay specificity and enabling detection of the variety of Neu5Gc-bearing N-glycans. By combining orthogonal dimensions of evidence, including chromatographic separation, isotopic distribution matching, and composition-confirming MS/MS, LC-MS confidently resolved Neu5Gc signals from noise, even at low abundance. In comparison, DMB-LC-FLR was limited to two orders of magnitude dynamic range, insufficient for detection of Neu5Gc in commercially available pooled human sera. These findings strongly support that DMB-LC-FLR assay specificity and sensitivity are insufficient for Neu5Gc detection in human samples due to noise overwhelming the Neu5Gc signal. By establishing a reusable benchmarking framework for future glycomic studies, we aim to use LC-MS to improve the measurement of Neu5Gc in clinical samples.

biochemistry↗

bioRxiv2026

Real-time mass defect-driven prediction of glycopeptide precursors enables enrichment-free serum glycoproteomics

Glycopeptide enrichment remains a cornerstone in glycoproteomics, but bias and reproducibility issues continue to hinder biological insight and clinical translation. Employing curated glycoproteomics datasets and machine learning, we trained a glycopeptide classifier to recognize N-glycopeptide precursors through mass defect signatures. Integration of the classifier into a data-dependent acquisition framework facilitated real-time prediction of N-glycopeptides from human serum and revealed sex differences in murine plasma fucosylation opening avenues for enrichment-free glycoproteomics.

bioinformatics↗

bioRxiv2026

Contextualised real-time mass spectrometry improves glycosylation detection and characterisation

Glycosylation is a structurally diverse, non-template-driven modification whose analysis by liquid chromatography-mass spectrometry is constrained by discovery-mode acquisition rules developed for proteomics. Data-dependent acquisition filters, such as intensity-based precursor selection and charge-state exclusion, map poorly onto glycan analysis, which span wide ranges of charge state and abundance independent of their biological importance. Here we present glycosylation real-time mass spectrometry (GlycoRTMS), an instrument-API method that annotates observed precursor masses with glycan compositions in real time and uses this context to guide fragmentation. Composition-aware precursor prioritisation sampled deeper into the precursor space, expanding MS2 coverage of a hyaluronic acid hydrolysate from four to eight oligosaccharide subunits. Charge-state-specific collision energy equations tailored to oligosaccharides produced complete fragment ladders where fixed normalised collision energy did not. MS3 triggering gated by both diagnostic ions and glycan composition matching enabled efficient, chromatography-compatible characterisation of O-acetylated sialic acids and identified product ions specific to O-acetylation. Together, these strategies improve both the depth and quality of glycan detection and characterisation within a single injection.

biochemistry↗

bioRxiv2026

GlyComboCLI enables command line-based FAIR workflows for glycan composition assignment in mass spectrometry data

Glycans are integral biomolecules whose presence cannot be predicted from genomic data alone, necessitating experimental characterisation through approaches including mass spectrometry. Assignment of glycan compositions to observed mass to charge ratios is computationally challenging due to the potential monosaccharide diversity and existing tools lack the required flexibility for integration into automated bioinformatic workflows. Here, we present GlyComboCLI, an open-source command-line application for the assignment of glycan compositions to mass spectrometry data which expands upon the previous GUI application, GlyCombo. GlyComboCLI accepts mass lists and vendor-neutral mzML files, supports diverse monosaccharides, derivatisation states, reducing-end modifications and adducts, and is validated through automated tests spanning supported search parameters, input formats, and instrument vendors. Outputs are compatible with downstream tools including Skyline and GlycoWorkBench while GlyTouCan accessions support persistent identification of registered base compositions. Deployment as a standalone executable, a Docker container, and a Galaxy tool, supports FAIR and reproducible workflows. Applied to published mouse and human glycomics datasets, GlyComboCLI reproduced major qualitative glycomic motifs and relative abundance trends, while Galaxy workflow reproduced expected effects of sialidase treatment. These results demonstrate a flexible, scalable, and reproducible approach for glycan composition assignment within automated glycomics workflows.

bioinformatics↗

bioRxiv2025

Anion supercharging enables structural assignment of oxidatively released O-glycans

O-glycan analysis faces analytical challenges due to poor fragmentation characteristics of oxidatively released O-glycan acids, which can preserve labile O-acetyl modifications but suffer from charge fixation at the reducing end. This study introduces a supercharging approach using hexafluoroisopropanol (HFIP) and butylamine mobile phase additives for enhanced mass spectrometric analysis of bleach-released O-glycan acids. HFIP/butylamine increased average charge states by up to 50% compared to traditional mobile phases, dramatically improving MS2 fragmentation quality and enabling discrimination between isomeric compositions and structures. Enhanced fragmentation enabled confident identification of sialic acid variants and acetylation patterns, overcoming the analytical bottleneck of poor O-glycan acid fragmentation. Computational optimization reduced database search times by up to ninefold while maintaining sensitivity. Application to multi-species gastric mucins revealed distinct glycosylation profiles. Porcine mucin showed predominant serine-linked neutral structures, while bovine and ovine mucins exhibited primarily threonine-linked sialylated glycans. A minor fraction of porcine and bovine mucins contained acetylated serine-linked sialylated glycans. This methodology provides a comprehensive framework for O-glycan characterization while preserving biological modifications.

biochemistry↗

bioRxiv2025

Native PGC-LC-MS Profiling Reveals Distinct O-Acetylation Patterns of Sialylated N-Glycans Across Mammalian Sera

O-acetylation of sialic acids represents an additional layer of structural diversity and biological complexity, occurring at various hydroxyl positions (commonly C-7, C-8, or C-9) of the sialic acid residue. This modification modulates the recognition of sialylated glycans by lectins, antibodies, and viral proteins, and contributes to viral tropism and host susceptibility, particularly in influenza and coronaviruses that bind O-acetylated sialylated receptors. However, current LC-MS glycomics workflows commonly employ reduction or permethylation, which, while improving chromatographic stability and ionisation, result in the loss of labile O-acetyl groups, obscuring their biological relevance. Native glycan analysis, in contrast, preserves the complete structural integrity of glycans, enabling accurate detection of labile modifications. Using a native released glycan workflow limited to pH [≤] 8, O-acetylated N-glycans were detected in mouse and rat sera that were previously undetectable under basic derivatisation conditions. Beam-type collision-induced dissociation generated the most informative fragmentation spectra, with diagnostic ions confirming O-acetylated NeuGc and NeuAc residues. Chromatographic profiling revealed later elution and broadened peak shapes for O-acetylated species, consistent with increased hydrophobicity and microheterogeneity. A checkpoint-based identification workflow incorporating isotopic, chromatographic, and MS2 criteria reduced false positives, retaining only 3-5% of putative O-acetylated glycans as confident identifications. Quantitative comparison across species revealed extensive O-acetylation in rat (53.4%) and moderate modification in mouse (8.8%), but none detectable in human serum. These findings establish a robust analytical framework for native detection and characterisation of O-acetylated N-glycans, revealing species-specific regulation of this labile modification.

biochemistry↗

bioRxiv2025

N-Glycopedia: Libraries for Native N-glycan Structural Analysis

Studies on glycans of glycoproteins are hampered by the lack of standards that reflect the wide diversity in structure typically observed. To this end we have exploited a large library of N-glycan standards comprised of a unique collection of 226 N-glycans including oligomannose, hybrid, and complex-type. We generated a method employing porous graphitized carbon (PGC) and liquid chromatography mass spectrometry (PGC-LC-MS), which can provide a high degree of resolution of underivatized N-glycan structures. Chromatogram libraries arising from these studies include retention time data, diagnostic fragments, and validated structural assignments, providing a robust platform for both targeted and discovery-based glycomics. We refer to this as an N-glycopedia, the first type of resource in which researchers can compare this collective data to N-glycans under study and overcome the limitations of only having compositional data and predicted structures. The technology is easily expandable to include additional N-glycans as new standards become available.

biochemistry↗

bioRxiv2024

Vibrio cholerae RbmB is an α-1,4-polysaccharide lyase with biofilm-dispersal activity against Vibrio Polysaccharide (VPS)

Many pathogenic bacteria form biofilms as a protective measure against environmental and host hazards. The underlying structure of the biofilm matrix consists of secreted macromolecules, usually including exopolysaccharides. To escape the biofilm, bacteria may produce a number of matrix-degrading enzymes, including glycosidic enzymes that digest exopolysaccharide scaffolds. The human pathogen Vibrio cholerae assembles and secretes an exopolysaccharide called VPS (Vibrio polysaccharide) which is essential in most cases for the formation of biofilms and consists of a repeating tetrasaccharide unit. Previous studies have indicated that a secreted glycosidase called RbmB is involved in Vibrio cholerae biofilm dispersal, although the mechanism by which this occurs is not understood. To approach the question of RbmB function, we recombinantly expressed and purified RbmB and tested its activity against purified VPS. Using a fluorescence-based biochemical assay, we show that RbmB specifically cleaves VPS in vitro under physiological conditions. Analysis of the cleavage products using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the -1,4 linkage between D-galactose and a modified L-guluronic acid) into a mixture of tetramers and octamers. We demonstrate that the product of the cleavage contains a double bond in the modified-guluronic acid ring, strongly suggesting that RbmB is cleaving using a glycoside lyase mechanism. Finally, we show that recombinant RbmB from Vibrio cholerae and the related aquatic species Vibrio coralliilyticus are both able to disperse living Vibrio cholerae biofilms. Our results support the role of RbmB as a polysaccharide lyase involved in biofilm dispersal as well as an additional glycolytic enzyme to add to the toolbox of potential therapeutic antibacterial enzymes. Author summaryBiofilms are protective sheaths produced by many bacteria that play important roles in survival in the environment and in hosts during infection. Understanding how biofilms form and disperse is essential in the fight against harmful bacterial pathogens. Vibrio cholerae is a historically significant human pathogen that forms biofilms made of a polysaccharide called VPS and secreted accessory proteins. Within the V. cholerae biofilm gene cluster is a gene encoding a protein called RbmB, which is known to play a role in biofilm dispersal. Using a combination of biochemical and analytical tools, we show that RbmB indeed cleaves VPS into 4- and 8-monosaccharide fragments at a unique site and demonstrate that it falls into the family of polysaccharide lyase enzymes (as opposed to the more common hydrolases). Furthermore, recombinant RbmB disperses living V. cholerae biofilms further indicating that it may play a role in VPS processing or degradation.

biochemistry↗

bioRxiv2020

Integrated glycoproteomics identifies a role of N-glycosylation and galectin-1 on myogenesis and muscle development

ABSTRACTMany cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsAAV adeno-associated virusANOVAanalysis of variationCBDscarbohydrate binding domainsCDGscongenital disorders of glycosylationCRISPRclustered regularly interspaced short palindromic repeatsCIDcollisional induced dissociationDTTdithiothreitolECMextracellular matrixEThcDelectron transfer dissociation with higher collisional dissociation supplemental activationFBSfetal bovine serumFDRfalse discovery rateFAformic acidGBPsglycan-binding proteinsHCDhigher collisional dissociationHILIChydrophilic interaction liquid chromatographyMCSmultiple cloning siteMeCNacetonitrileNMJneuromuscular junctionsPCAprincipal component analysisPGCporous graphitized carbonPSMpeptide spectral matchTEABtriethylammonium bicarbonateTFAtrifluoroacetic acidTMTtandem mass tagsView Full Text

biochemistry↗

bioRxiv2019

Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes

Cell surface glycoproteins play critical roles in maintaining cardiac structure and function in health and disease and the glycan-moiety attached to the protein is critical for proper protein folding, stability and signaling. However, despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. In the current study, we used porous graphitized carbon liquid chromatography interfaced with mass spectrometry (PGC-LC-MS) to generate glycan structure libraries for primary human heart tissue homogenate, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM). Altogether, we established the first reference structure libraries of the cardiac glycome containing 265 N- and O-glycans. Comparing the N-glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, 21 structures significantly differed, and the high mannose class is increased in enriched CM. Moreover, by comparing primary CM to hiPSC-CM collected during 20-100 days of differentiation, dynamic changes in the glycan profile throughout in vitro differentiation were observed and differences between primary and hiPSC-CM were revealed. Namely, >30% of the N-glycome significantly changed across these time-points of differentiation and only 23% of the N-glycan structures were shared between hiPSC-CM and primary CM. These observations are an important complement to current genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. Finally, these data are expected to support future regenerative medicine efforts by informing targets for evaluating the immunogenic potential of hiPSC-CM and harnessing differences between immature, proliferative hiPSC-CM and adult primary CM.

biochemistry↗

bioRxiv2018

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

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

bioinformatics↗