As an essential modification, O-linked β-N-acetylglucosamine (O-GlcNAc) modulates the functions of many proteins. However, site-specific characterization of O-GlcNAcylated proteins remains challenging. Herein, an innovative material grafted with nitro-oxide (N→O) groups was designed for high affinity enrichment for O-GlcNAc peptides from native proteins. By testing with synthetic O-GlcNAc peptides and standard proteins, the synthesized material exhibited high affinity and selectivity. Based on the material prepared, we developed a workflow for site-specific analysis of O-GlcNAcylated proteins in complex samples. We performed O-GlcNAc proteomics with the PANC-1 cell line, a representative model for pancreatic ductal adenocarcinoma. In total 364 O-GlcNAc peptides from 267 proteins were identified from PANC-1 cells. Among them, 183 proteins were newly found to be O-GlcNAcylated in humans (with 197 O-GlcNAc sites newly reported). The materials and methods can be facilely applied for site-specific O-GlcNAc proteomics in other complex samples.
Keywords: O-GlcNAc; O-GlcNAcylation; glycopeptide enrichment; glycoproteomics; hydrogen bonding interaction; nitro-oxide-grafted nanosphere.