Endo‐M Mediated Chemoenzymatic Approach Enables Reversible Glycopeptide Labeling for O‐GlcNAcylation Analysis

Abstract

To selectively enrich O-linked β-N-acetylglucosamine (O-GlcNAc) peptides in their original form from complex samples, we report the first reversible chemoenzymatic labeling approach for proteomic analysis. In this strategy, the O-GlcNAc moieties are ligated with long N-glycans using an Endo-M mutant, which enables the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). The attached glycans on the enriched glycopeptides are removed by wild-type Endo-M/S to restore the O-GlcNAc moiety. Compared with classic chemoenzymatic labeling, this approach enables the tag-free identification, and eliminates the interference of bulky tags in glycopeptide detection. This approach presents a unique avenue for the proteome-wide analysis of protein O-GlcNAcylation to promote its mechanism research.

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis

“…Post‐translational modifications (PTMs), whose expression patterns and modification sites have attracted much attention, is a process that endows proteins with versatility. As the most widespread and essential PTMs in cells, protein phosphorylation and glycosylation play critical regulatory roles in cell cycle, growth, apoptosis, and signal transduction [1–3]. In addition, the occurrence and development of various diseases, including blood diseases, genetic diseases, lung diseases, neurodegenerative diseases, and cancer, are tightly related to the abnormal phosphorylation and glycosylation of proteins [4–6].…”

Multi-comparative Thermal Proteome Profiling Uncovers New O-GlcNAc Proteins in a System-wide Method

“…Due to the low stoichiometry as well as the reversibility of O-GlcNAcylation, much progress has been made in related enrichment approaches prior to liquid-chromatography tandem mass spectrometry (LC-MS/MS), such as in lectin affinity or hydrophilic interaction liquid chromatography (HILIC) capture. , Meanwhile, numerous UDP-GlcNAc derivatization-based enrichment techniques like chemoenzymatic and metabolic labeling methods have also been developed. − With such powerful enrichment approaches, thousands of O-GlcNAc proteins have been successfully explored with sites identified. Meanwhile, it is worth noting that insufficient binding affinity might lead to false positives and complex derivatization might induce unexpected labeling. , Protein/peptide microarray-based methods have also been applied but are disadvantaged from the low specificity of the anti-O-GlcNAc antibody and finite target candidates .…”