O‑Glycopeptide Truncation Strategy for Heterogeneous O‑GalNAc Glycoproteomics Characterization

• Published in: Analytical chemistry (Washington) Vol. 95; no. 26; pp. 10017 - 10024
• Main Authors: Liu, Luyao, Zhu, He, Liu, Lei, You, Xin, Mao, Jiawei, Wang, Yan, Liu, Xiaoyan, Qin, Hongqiang, Dong, Mingming, Ye, Mingliang
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.07.2023
• Subjects: Biological activity; Biological properties; Biological samples; Chemistry; Enrichment; Glycan; Glycopeptides; Glycopeptides - analysis; Glycoproteins; Glycoproteins - chemistry; Glycosylation; Humans; Mass spectrometry; Mass spectroscopy; Peptides; Polysaccharides; Polysaccharides - analysis; Proteins; Proteolysis; Proteomics - methods; Sialic acids
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.3c01327

Mucin-type O-glycosylation (or O-GalNAcylation) takes place on most membrane and secretory proteins and is vital in regulating protein functions and many biological processes. O-GalNAcylation generally exhibits highly diverse and dense O-glycans linked to carrier proteins, which challenges the analysis of O-GalNAc glycoproteome using conventional methodologies. Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes proteases or O-glycopeptidases for targeted cleavage of the enriched tryptic O-glycopeptides. It simplifies the O-glycopeptide backbones, O-glycans, or both, and has been shown to aid the improvement of the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. The enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for mass spectrometry detection and database search in general bottom-up glycoproteomics. We also investigate different proteolysis which could be well integrated into the O-glycopeptide truncation strategy. For large-scale analysis, we exploit different truncation schemes and identify nearly 2000 O-glycopeptides corresponding to 391 glycoproteins from 75 μL human serum, achieving the deepest-scale coverage of O-glycoproteins compared to other plasma/serum O-glycoproteomic studies. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.