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Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies for Glycopeptide Analysis

ABSTRACT: Mass spectrometry-based glycoproteomics studies now routinely report thousands of intact glycopeptides. Diverse informatics solutions aiding the challenging glycopeptide identification process have recently appeared, but their strengths and weaknesses have not been established leaving a critical knowledge gap that prevents rapid progress in the field. This interlaboratory study comprising both expert users (13 teams) and developers (9 teams) of glycoproteomics software is the first to evaluate the relative performance of current informatics solutions for accurate and comprehensive N- and O-glycopeptide identification. Two LC-MS/MS datasets of intact glycopeptides from serum proteins were shared with all teams. The relative team performance for large-scale glycopeptide analysis was systematically established through 11 orthogonal performance tests based on the assigned spectra reported by each team. This study has found that a diversity of approaches exists for comprehensive glycopeptide data analysis, has demonstrated the team-to-team (vari)ability of identifying intact glycopeptides from shared datasets, and, importantly, has revealed several high-performance informatics solutions and search strategies that will be useful to improve the challenging glycoproteomics data analysis in the immediate future.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

SUBMITTER: Rebeca Kawahara

LAB HEAD: Morten Thaysen-Andersen

PROVIDER: PXD024101 | Pride | 2021-11-03

REPOSITORIES: Pride

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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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Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies for Glycopeptide Analysis

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