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On-tissue spatially-resolved glycoproteomics guided by N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape

ABSTRACT: Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan MSI and spatially-resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, making our study the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially-resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Canis Familiaris (dog) (canis Lupus Familiaris)

TISSUE(S): Brain

DISEASE(S): Brain Cancer

SUBMITTER: Stacy Malaker

LAB HEAD: Stacy Malaker

PROVIDER: PXD025537 | Pride | 2022-05-04

REPOSITORIES: Pride

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On-tissue spatially-resolved glycoproteomics guided by N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape

Project description:Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan MSI and spatially-resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, making our study the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially-resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.

2023-03-11 | PXD025826 | Pride

Glycan shield of the ebolavirus envelope glycoprotein GP

Project description:The envelope glycoprotein GP of the ebolaviruses is essential for host cell attachment and entry. It is also the primary target of the protective and neutralizing antibody response in both natural infection and vaccination. GP is heavily glycosylated with up to 17 predicted N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation of GP is important for host cell attachment to cell-surface lectins, as well as GP stability and fusion activity. Moreover, it has been shown to shield GP from neutralizing activity of serum antibodies. Here, we use mass spectrometry-based glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP, including N-, O-, and C-linked glycans.

2022-08-02 | PXD031459 | Pride

HCOV-NL63 spike protein glycopeptides

Project description:HCoV-NL63 is a coronavirus that can cause severe lower respiratory tract infections requiring hospitalization. Of great interest is understanding the HCoV-NL63 coronavirus spike glycoprotein trimer, which is the conformational machine responsible for entry into host cells and the sole target of neutralizing antibodies during infection. We utilized an electron-transfer/higher energy collision dissociation ion fragmentation scheme (Frese, C. K. et al., 2013.) in combination with cryo-electron microscopy to resolve the extensive glycan shield that obstructs the protein surface. These glycans provide a structural framework to understanding the accessibility of the protein to antibodies.

2016-09-27 | PXD004557 | Pride

O-linked glicoproteomics of gastric cancer cell lines OCUM-1, N87 and KATO-III

Project description:Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, continuously requiring innovative target therapies. The sialyl-lewis A (SLeA) antigen offers tremendous potential for precise cancer targeting. This glycan is rarely expressed by healthy tissues and blood cells but is often present in cancer cells of high metastatic potential as well as in the metastasis. To improve further on the gastric cancer nature of this glycan we have zoomed in on the SLeA-glycoproteome envisaging the identification of possible targetable biomarkers. SLeA-glycoproteins were isolated by immunoprecipitation from well-known cell models of gastric cancer reflecting different histological subtypes of the disease (KATO-III, OCUM-1 and N87 cells). Glycoproteins with affinity for E-selectin were also isolated and analyzed. Proteomics study employed a typical nanoLC-ESI-Orbitrap-MS/MS platform. The well-known molecular heterogeneity of these cell lines was reflected in the markedly different glycoproteomics signatures observed. Nevertheless, a restricted panel of SLeA-expressing glycoproteins with affinity for E-selectin remained conserved between the cell lines, consisting of a promising list of potentially targetable biomarkers for bladder cancer.

2020-05-11 | PXD016968 | Pride

Evaluation and Optimization of High-Field Asymmetric Waveform Ion Mobility Spectrometry for Multiplexed Quantitative Site-specific N-glycoproteomics

Project description:The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified IgM protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass-spectrometry-based qualitative and quantitative N-glycoproteomics.

2021-05-05 | PXD023951 | Pride

Expression of glycan receptors for influenza A viruses in mallards, chickens, and tufted ducks

Project description:Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Investigating the molecular basis for virus–host interactions enabling this process is vital to understand zoonotic IAV spread. Receptor incompatibility has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative receptors for IAV in mallards, chickens and tufted ducks; three bird species with different roles in the zoonotic ecology of IAV. The methodology used could not only pinpoint specific glycan structures to the specific glycosylation sites of identified glycoproteins but could also be used to successfully discriminate α2,3- from α2,6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides during MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex α2,3 and α2,6-linked Neu5Ac N-glycans including α2,3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2,3 and α2,6-linked Neu5Ac. Furthermore, we reveal many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures previously anticipated to be mammalian specific in all three bird species have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.

2024-04-18 | PXD036263 | Pride

Discovery of a pathway for endogenous mucin glycodomain catabolism in mammals

Project description:Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N-glycoconjugates in human lysosomal storage disorders, is that proteolysis and glycan degradation occur largely independently. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O-glycosylated mucin domains, without prior deglycosylation. Using activity screening coupled with mass spectrometry we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis resulted in accumulation of mucins in liver-resident macrophages. Our findings suggest that mucin-degrading activity is not limited to the bacterial kingdom and is a component of glycoprotein catabolism in mammalian tissues.

2022-09-03 | PXD027616 | Pride

Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC)

Project description:A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography (HILIC). The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg+2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly-hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity between 6- to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, N- and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with “crosstalk” modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase (RP) LC-MS/MS analysis. Between 47-100% of the peptides with more than one phosphate or sialyl- residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.

2021-06-01 | PXD022988 | Pride

Analysis of Pancreatic Extracellular Matrix Protein Post-Translational Modifications via Electrostatic Repulsion-Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry

Project description:The pancreas is a vital organ with digestive and endocrine roles, and diseases of the pancreas affect millions of people yearly. A better understanding of the pancreas proteome and its dynamic post-translational modifications (PTMs) is necessary to engineer higher fidelity tissue analogues for use in transplantation. The extracellular matrix (ECM) has major roles in binding and signaling essential to the viability of insulin-producing islets of Langerhans. To analyze post-translational modifications (PTMs) in the pancreas, native and decellularized tissues from four donors were analyzed.

2021-08-06 | PXD025048 | Pride

Re-analysis of glycoproteomics data with MSFragger-Glyco

Project description:Reanalysis of submissions PXD011533 and PXD009476 using MSFragger-Glyco mode. Processed MSFragger results files (pepXML) and PSM tables (psm.tsv) supporting MSFragger-Glyco manuscript (Fast and Comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco. Recent advances in methods for enrichment and mass spectrometric analysis of intact glycopeptides have produced large-scale glycoproteomics datasets, but interpreting this data remains challenging. We present MSFragger-Glyco, aa new glycoproteomics mode of the MSFragger search engine, called MSFragger-Glyco, for fast and sensitive identification of N- and O-linked glycopeptides and open glycan searches. Reanalysis of recent N-glycoproteomics data resulted in annotation of 80% more glycopeptide-spectrum matches (glycoPSMs) than previously reported. In published O-glycoproteomics data, our method more than doubled the number of glycoPSMs annotated when searching the same glycans as the original search and yielded 4-6-fold increases when expanding searches to include additional glycan compositions and other modifications. Expanded searches also revealed many sulfated and complex glycans that remained hidden to the original search.

2020-10-06 | PXD021196 | Pride

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