Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry

Zihao Wang,Namrata D Udeshi,Meaghan O'Malley,Jeffrey Shabanowitz,Donald F Hunt,Gerald W Hart

doi:10.1074/mcp.m900268-mcp200

Zihao Wang, Namrata D Udeshi + Show 4 more

Open Access

https://doi.org/10.1074/mcp.m900268-mcp200

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Abstract

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

Highlights

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked ␤-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins
Problems associated with the identification of O-GlcNAc sites include the following. (a) O-GlcNAc is quickly removed by hydrolases during cell lysis. (b) Like phosphorylation, OGlcNAc is usually present in less than stoichiometric amounts at given sites on protein substrates. (c) O-GlcNAc is readily lost as an oxonium ion during conventional peptide sequence analysis by collision-activated dissociation (CAD). (d) Modified and unmodified forms of the peptide often co-elute during reverse phase HPLC, and the preferential ionization of the unmodified peptide suppresses the signal observed for the corresponding O-GlcNAc-modified peptide
O-GlcNAc-modified peptides were biotinylated with hydrazine chemistry, isolated on a column packed with avidin beads, eluted with free biotin, and sequenced by ETD mass spectrometry

Summary

Introduction

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked ␤-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. O-GlcNAc-modified peptides were biotinylated with hydrazine chemistry, isolated on a column packed with avidin beads, eluted with free biotin, and sequenced by ETD mass spectrometry.

Results

Conclusion