Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chrormatography-tandem mass spectrometry

P. A. Haynes*, R. Aebersold

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

61 Citations (Scopus)

Abstract

Glycoproteins carrying O-linked N-acetylglucosamine (O-GlcNAc) modifications have been isolated from a wide range of organisms ranging from trypanosomes to humans. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli. Concurrent advances in biological mass spectrometry (MS) have facilitated high-sensitivity protein identification by tandem MS. In this study, we show that the lability of the O-GlcNAc moiety to low-energy collision in tandem MS offers a means of distinguishing such peptides from others that are not modified. The differential between the energy required to remove the O-GlcNAc group and the energy required to fragment the peptide chain allows the O-GlcNAc group to be detected and the peptide sequence, and therefore the protein, to be identified. This technique thus allows the simultaneous detection and identification of O-GlcNAc-modified peptides, even when present at low levels in complex mixtures. The method was initially developed and validated using a synthetic O-GlcNAc-modified peptide and then applied to the detection of an extremely low abundance O-GlcNAc-modified peptide from bovine α-crystallin. We believe that with further development this assay system may prove to be a useful tool for the direct investigation of intracellular O-GlcNAc levels, thus providing valuable insights into the physiological role of O-GlcNAc modified proteins.

Original languageEnglish
Pages (from-to)5402-5410
Number of pages9
JournalAnalytical Chemistry
Volume72
Issue number21
DOIs
Publication statusPublished - 1 Nov 2000
Externally publishedYes

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