Dataset Information

A comprehensive, open-source platform for mass spectrometry based glycoproteomics data analysis

ABSTRACT: Glycosylation is among the most abundant and diverse protein post-translational modifications (PTMs) identified to date. The structural analysis of this PTM is challenging due to the diverse monosaccharides which are not conserved among organisms, the branched nature of glycans, their isomeric structures, and heterogeneity in the glycan distribution at a given site. Glycoproteomics experiments have adopted the traditional high-throughput LC-MSn proteomics workflow to analyze site-specific glycosylation. However, comprehensive computational platforms for data analyses are scarce. To address this limitation, we present a comprehensive, open-source, modular software for glycoproteomics data analysis called GlycoPAT (GlycoProteomics Analysis Toolbox). The program introduces ‘SmallGlyPep’ as a minimal linear representation of glycopeptides for MSn data analysis. It enables MS/MS analysis of N- and O-linked glycosylation using a novel scoring scheme to rank the hits based on cross-correlation and probability based analysis. False discovery calculations, parallel computing facilities and user-friendly GUIs (Graphical User Interfaces) are also provided. GlycoPAT is used to analyze site-specific glycosylation on simple glycoproteins, protein mixtures and human plasma cryoprecipitate. The results show that the simultaneous consideration of peptide and glycan fragmentation enables high quality MS spectrum annotation in three common MS/MS fragmentation modes: CID, HCD and ETD.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Kai Cheng

LAB HEAD: Sriram Neelamegham

PROVIDER: PXD006031 | Pride | 2017-09-19

REPOSITORIES: Pride

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A Comprehensive, Open-source Platform for Mass Spectrometry-based Glycoproteomics Data Analysis.

Liu Gang G Cheng Kai K Lo Chi Y CY Li Jun J Qu Jun J Neelamegham Sriram S

Molecular & cellular proteomics : MCP 20170908 11

Glycosylation is among the most abundant and diverse protein post-translational modifications (PTMs) identified to date. The structural analysis of this PTM is challenging because of the diverse monosaccharides which are not conserved among organisms, the branched nature of glycans, their isomeric structures, and heterogeneity in the glycan distribution at a given site. Glycoproteomics experiments have adopted the traditional high-throughput LC-MS<sup>n</sup> proteomics workflow to analyze site- ...[more]

PMID: 28887379

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Project description:Neutrophils are short-lived yet vital innate immune cells equipped with bioactive glycoproteins packed in cytosolic granules that can readily be mobilised to elicit an effective and timely response against invading pathogens. Since the dynamic glycosylation processes underpinning the dramatic metamorphosis associated with myeloid progenitor-to-neutrophil differentiation remain unmapped, we here perform a comprehensive spatiotemporal profiling of the complex N-glycoproteome of isolated granule populations from blood-derived neutrophils and during maturation of bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Firstly, glycomics indicated that the granule populations of mature (resting) neutrophils exhibit distinctive glycophenotypes including, most strikingly, unusual paucimannosidic- and monoantennary complex-type N-glycans in azurophilic granules. The granule-specific N-glycosylation features were recapitulated by glycoproteomics, which also uncovered extensive site- and protein-specific N-glycosylation decorating 4,772 N-glycopeptides from 352 N-glycoproteins across the neutrophil granules. Excitingly, comprehensive mining of proteomics and transcriptomics data collected from discrete myeloid progenitor stages for glycopeptide and glyco-enzyme expression revealed that dramatic glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven predominantly by proteome expression changes rather than modulation of the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly reduced with myeloid differentiation leading to reduced N-glycosylation efficiency in late-stage neutrophil maturation and in corresponding granules. Our study provides new spatiotemporal insights into the dynamic neutrophil N-glycoproteome, which exhibits intriguingly complex site-, protein- and granule-specific N-glycosylation features and which undergoes strong remodelling during myeloid progenitor-to-neutrophil metamorphosis.

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