The reversible chemoenzymatic labeling strategy allows for an in-depth analysis of protein O-GlcNAcylation

O-Linked β-N-acetylglucosamine (O-GlcNAcylation), an important post-translational modification (PTM) of proteins, is involved in diverse biological functions.

Reversible modification of O-GlcNAc confers on-off protein functions during biological processes. Aberrations in O-GlcNAcylation are closely associated with many metabolic diseases along with the invasion and metastasis of several tumors.

Recently, a research team led by Prof. Ye Mingliang and Prof. Qin Hongqiang from the Dalian Institute of Chemical Physics (DICP) of the Chinese Academy of Sciences (CAS), in collaboration with Prof. Huang Wei from the Shanghai Institute of Materia Medica of CAS, developed a new strategy of reversible chemoenzymatic labeling of O-GlcNAc glycopeptides, which enabled in-depth analysis of protein O-GlcNAcylation.

Their results were published in applied Chemistry on March 14th.

To enable proteome-wide analysis of O-GlcNAcylation, it is essential to selectively enrich for glycopeptides from digests of complex samples.

Many researchers have aimed at enriching O-GlcNAcylated peptides prior to analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, most approaches suffer from weak binding affinity or bulky tags that interfere with the enrichment and identification of O-GlcNAcylated peptides.

In this newly developed strategy, the O-GlcNAc moieties were ligated to long N-glycans using an Endo-M mutant, allowing for the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). Then the attached glycans on the enriched glycopeptides were removed by wild-type endo-M/S to regenerate the O-GlcNAc moiety.

Compared to classical chemoenzymatic labeling, this approach enabled tag-free identification and eliminated the interference of bulky tags in glycopeptide detection.

Furthermore, the researchers identified 657 potential O-GlcNAc glycosites from only 0.4 mg of HeLa nuclear proteins using this method, which required only 1/10 of the protein samples for a comparable O-GlcNAcylation analysis, indicating the high sensitivity of the method.

Overall, they identified 1,414 glycosites from only 1.1 mg protein samples, and 45% of them were not present in O-GlcNAcAltas of all human samples over the past 35 years, improving analysis coverage of protein O-GlcNAcylation.

“This tag-free enrichment strategy represents a unique avenue for proteome-wide analysis of O-GlcNAcylation and promotes mechanistic studies,” said Prof. Ye.