Biology subjects

Packer, N. H.

Publications and source records attributed to Packer, N. H..

7 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

Immobilised enzyme reactors for post-production glycan modification of purified glycoproteins

Controlling protein glycosylation as a critical quality attribute of biopharmaceuticals remains challenging when glycosylation is coupled to cellular production systems. Here, we present a proof-of-concept glycosyltransferase immobilised enzyme reactor (IMER) housed within a 3D-printed column that enables directed post-production glycan modification of purified glycoproteins. Using {beta}-1,4-galactosyltransferase ({beta}4GalT1-IMER) and -2,6-sialyltransferase (ST6Gal1-IMER) immobilised on Ni-NTA resin, the IMER achieved near-complete galactosylation and substantial sialylation of partially deglycosylated bovine fetuin N-glycans with their respective substrates with a maximum substrate-enzyme contact time of four minutes. Isomeric-level analysis revealed arm-specific addition preferences for both enzymes, consistent with known specificities. The modular IMER design permits sequential connection of individual enzyme chambers, potentially offering a scalable, plug-and-play platform for constructing defined glycan structures on recombinant glycoprotein therapeutics.

molecular biology↗

bioRxiv2026

De-N-glycosylation of in vivo and in vitro adipogenic stem cell products unmasks differential expression of CD36 glycoprotein in human adipogenesis

Adipogenesis is the process of adipose-derived stem cells (ADSCs) responding to extracellular signals from the stem cell niche to differentiate into adipocytes (fat cells) and may be studied in vitro using a cocktail of chemicals that promote adipogenic differentiation to produce differentiated ADSCs (dADSCs). The global membrane N- and O-glycosylation changes of this process have been previously analysed and compared to native adipocytes as a benchmark for a true adipocyte profile, and revealed that bisecting GlcNAc type N-glycans are characteristic of adipogenesis. As stem cell differentiation has been widely reported to result in cellular protein changes, the same cells (ADSCs, dADSCs and mature adipocytes) were characterised for their membrane proteome here using label-free quantitative shotgun proteomics analysis. The membrane proteome displayed more differences in protein numbers between the cell types compared to the previously reported N-glycome which had shown high identical glycomes between stem cells and in vitro dADSCs, suggesting that the proteome is more dynamic during in vitro adipogenesis. Following the global shotgun proteomics analysis, a more targeted approach of carrying out proteomic analysis of de-N-glycosylated peptides of gel-separated proteins unearthed new glycoproteins not detected in the shotgun proteomic analysis. This approach identified the adipogenic marker, CD36, to be under-represented in the shotgun proteome analysis, but as the dominant (glyco)protein in the adipocyte membrane proteome that was also up-regulated at the mRNA transcript level in both the in vitro differentiated ADSCs (7.1-fold increase) and mature adipocytes (102.9-fold increase). A comparison of CD36 sequence coverage in the global shotgun analysis with the de-N-glycosylated CD36 revealed a 41% increase when N-glycans were removed prior to trypsin digestion, explaining its observed increased abundance and highlights the crucial need for de-N-glycosylation of proteins in proteomics experiments for increased identification of glycoproteins. The systems glycobiology approach by the integration of previously reported glycomics data and the proteomics and transcriptomics analyses in this work extended the investigation of membrane protein glycosylation changes in adipose-derived stem cell differentiation. The work provides a framework for future glycoproteomics-based investigations into the differentiation of stem cells into adipocytes, and will allow their related pathologies and potential therapeutic applications to be discovered. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/722121v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@189a786org.highwire.dtl.DTLVardef@5563b8org.highwire.dtl.DTLVardef@5cb5borg.highwire.dtl.DTLVardef@69e11f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

The molecular basis of immunosuppression by soluble CD52 is defined by interactions of N-linked and O-linked glycans with HMGB1 Box B

AbstractHuman soluble CD52 is a short glycopeptide comprising 12 amino acids (GQNDTSQTSSPS) which functions as an immune regulator by sequestering the pro-inflammatory high mobility group box protein 1 (HMGB1) and supressing immune responses. Recombinant CD52 has been shown to act as a broad anti-inflammatory agent, dampening both adaptive and innate immune responses. This short glycopeptide is heavily glycosylated, with a complex sialylated N-linked glycan at N3 and reported O- linked glycosylation possible on several serine and threonine residues. Previously we demonstrated that specific glycosylation features of CD52 are essential for its immunosuppressive function, with terminal -2,3-linked sialic acids required for binding to the inhibitory SIGLEC-10 receptor leading to T-cell suppression. Using high resolution mass spectrometry, we have further characterised the N- and O-linked glycosylation of Expi293 recombinantly produced CD52 at a glycopeptide and released glycan level, accurately determining glycan heterogeneity of both N- and O-linked glycosylation, and localising the site of O-glycosylation to T8 with high confidence and direct spectral evidence. This detailed knowledge of CD52 glycosylation informed the construction of a model system, which we analysed by molecular dynamics simulations to understand the mechanism of recognition and define interactions between bioactive CD52, HMGB1 and the SIGLEC-10 receptor. Our results confirm the essential role of glycosylation, more specifically hyper-sialylation, in the function of CD52, and identify at the atomistic level specific interactions between CD52 glycans and the Box B domain of HMGB1 that determine recognition, and the stability of the CD52/HMGB1 complex. These insights will inform the development of synthetic CD52 as an immunotherapeutic agent.

molecular biology↗

bioRxiv2022

Intermittent fasting has a diet-specific impact on the gut microbiota and colonic mucin O-glycosylation of mice

The colonic mucus layer and microbiota adhered to it are vital for mediating host metabolic, immune, and gut health. Yet, how intermittent fasting impacts these microbial communities and O-glycosylation of mucin proteins, the predominant component of the colonic mucus layer, remains largely unexplored. Here, using a C57BL/6J mouse model fed either a high-fat diet or normal chow, we examined the impact of a two-day a week fasting regimen on host physiology, faecal and colonic mucosal microbiota, and mucin O-glycosylation. Our results demonstrated distinct diet-specific impacts of intermittent fasting on host physiology; mice fed the high-fat diet had a lower body weight and improved glucose tolerance upon fasting, whilst there were no significant changes in mice fed the normal chow. This was observed despite the similar feed and energy intake between groups with and without fasting. There were significant changes in the faecal and colonic mucosal microbiota community structure and composition, and mucin O-glycosylation upon fasting in both dietary groups, but the specific nature of these alterations was diet-dependent. Correlation analysis revealed significant associations between fasting-mediated changes in the abundance of specific mucosal bacteria and O-glycan structures. While intermittent fasting is a popular means of extending healthy life expectancy, there is a lack of information on its impacts on the mucosal microbiota and colonic mucus layer, which are key determinants of gut health. Our study addresses this knowledge gap and serves as the first report on how intermittent fasting influences colonic mucin O-glycosylation and the associations between mucosal glycans and bacteria.

microbiology↗

bioRxiv2021

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

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

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↗