Mapping of O-glycopeptides Using EXoO and a Novel Data Search Workflow
Embargo until
2022-05-01
Date
2020-05-12
Authors
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Publisher
Johns Hopkins University
Abstract
Protein glycosylation is indisputably the most omnipresent protein modifications across mammalian cells. In eukaryotes, different types of oligosaccharides are linked to glycoproteins through two major types of glyosidic linkages, N- and O-linked glycosylation. However, the investigations on O-linked glycosylation has long been barricaded by the limitations on suitable methodologies. Recently, the development of chemical enzymatic method using solid phase peptide capture and specific releasing of O-glycosylated peptides followed by mass spectrometry analysis serves as the stepping stone for site-specific extraction of O-linked glycopeptide (EXoO)1. In this study, a novel data search workflow was adopted in addition to the incorporation of EXoO and liquid chromatography tandem mass spectrometry platform (LC-MS/MS). SEQUEST analysis assigned 19,275 spectra to 3,540 intact O-linked glycopeptides containing at least 2,600 O-linked glycosylation sites from 754 glycoproteins in human brain. Compared to 2,076 in kidney, 1,421 in T cells, and 805 in serum, human brain EXoO analysis identified the most O-linked glycopeptides and 1,206 were novel O-linked glycosylation sites, bringing the total identified O-linked glycopeptides to 4,672. Spectra were further analyzed using GPQuest, identifying at least 1,873 O-linked glycosylation sites on over 570 proteins with 61 O-glycan compositions in human brain tissues.
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Keywords
Proteomics, Protein glycosylation, Tandem mass spectrometry (LC-MS/MS), Site-specific Extraction of O-linked glycopeptides (EXoO), O-linked glycopeptide, O-glycoproteome, O-glycan, Intact glycopeptide, Data-dependent Analysis (DDA), Database search