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A Combinatorial Enzymatic Approach for Identifying O-glycopeptides

ABSTRACT: Proteins glycosylation is primarily characterized as N-glycosylation or O-glycosylation. Recognition of the consensus N-glycosylation sequon (N-X-S/T/C) has enabled the mapping of the glycosite occupancy of intact glycopeptides, whereas O-glycosylation consensus sequons do not exit, making the characterization of O-glycosites challenging because of the different degrees of occupancy within a protein. Most O-glycosylation occurs via O-GalNAcylation, which is critical to diverse biological functions. However, only a small fraction of the O-glycosites and their glycan structures have been identified. We introduced a robust and reliable O-glycoproteomic workflow to achieve the long-standing goal of glycobiology—identifying novel O-glycosylation profiles on a large-scale. We developed and implemented a novel O-glycoproteomic workflow through the use of complementary digestion with O-glycoprotease (IMPa), trypsin, Asp-N, and Glu-C for enrichment of O-glycopeptides. The N-terminal of O-glycosylated serine or threonine residues cleaved using IMPa (95% of total PSMs) and over 99% of O-glycopeptides were enriched by the RAX column. Our O-glycoproteomic workflow involving peptide identification (using sceHCD-based MS/MS) and annotation (using FDR evaluation) enabled the identification of O-glycosylation of 189 O-glycosites carrying 379 glycan structures on 79 O-glycoproteins in human plasma. Of the O-glycan forms, 91.7% were distinctively and abundantly observed in core 1 and 2 structures. Importantly, we assessed five well-characterized native proteins purified from human plasma (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) to validate the identification of O-glycosites with high confidence and confirm the presence of numerous novel glycosites. We believe that this combinatorial approach is broadly applicable for comprehensive characterization of O-glycoproteomes in biomedical research.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD042701 | Pride | 2024-04-25

REPOSITORIES: Pride

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			Action	DRS
	20230224_TK_O-glycoproteome_APOE-OG-Try_1.raw	Raw
	20230224_TK_O-glycoproteome_APOE-OG_1.raw	Raw
	20230224_TK_O-glycoproteome_FGA-FGB-OG-Glu.raw	Raw
	20230224_TK_O-glycoproteome_FGA-FGB-OG-Glu_1.raw	Raw
	20230224_TK_O-glycoproteome_FGA-FGB-OG-Glu_2.raw	Raw

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A Combinatorial Enzymatic Approach for Identifying O-glycopeptides

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