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Re-analysis of glycoproteomics data with MSFragger-Glyco

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Brain, T Cell, Blood Serum, Kidney

SUBMITTER: Daniel Polasky

LAB HEAD: Alexey Nesvizhskii

PROVIDER: PXD021196 | Pride | 2020-10-06

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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Re-analysis of glycoproteomics data with MSFragger-Glyco

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