Novel Antibodies for the Simple and Efficient Enrichment of Native O-GlcNAc Modified Peptides

Mol Cell Proteomics. 2021;20:100167. doi: 10.1016/j.mcpro.2021.100167. Epub 2021 Oct 20.

Abstract

Antibodies against posttranslational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of these PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1300 unique O-GlcNAc-modified peptides and over 1000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.

Keywords: HCD product-dependent ETD; O-GlcNAc; enrichment; glycoproteomics; oxonium ion ratios.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Brain
  • Glycopeptides / immunology*
  • Mice
  • Mouse Embryonic Stem Cells
  • N-Acetylglucosaminyltransferases / immunology*

Substances

  • Antibodies, Monoclonal
  • Glycopeptides
  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase