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

Rajan A Burt; Borislav Dejanovic; Hayley J Peckham; Kimberly A Lee; Xiang Li; Johain R Ounadjela; Anjana Rao; Stacy A Malaker; Steven A Carr; Samuel A Myers

doi:10.1016/j.mcpro.2021.100167

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.

Authors

Affiliations

¹ The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
² Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
³ Cell Signaling Technology, Inc, Danvers, Massachusetts, USA.
⁴ La Jolla Institute for Immunology, La Jolla, California, USA.
⁵ La Jolla Institute for Immunology, La Jolla, California, USA; Department of Pharmacology, University of California San Diego, La Jolla, California, USA; Moores Cancer Center, University of California San Diego, La Jolla, California, USA.
⁶ Department of Chemistry, Yale University, New Haven, Connecticut, USA.
⁷ The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address: scarr@broad.mit.edu.
⁸ The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; La Jolla Institute for Immunology, La Jolla, California, USA. Electronic address: sam@lji.org.

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

Abstract

Publication types

MeSH terms

Substances

Grants and funding