Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 22, Issue 5, Pages 931-942Publisher
SPRINGER
DOI: 10.1007/s13361-011-0107-y
Keywords
Tandem mass spectrometry; O-GlcNAc; Pulsed Q dissociation; Electron-transfer dissociation
Funding
- Studienstiftung des deutschen Volkes e. V.
- Faculty Graduate Center Weihenstephan of TUM Graduate School at the Technische Universitat Munchen, Germany
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The modification of serine and threonine residues in proteins by a single N-acetylglucosamine (O-GlcNAc) residue is an emerging post-translational modification (PTM) with broad biological implications. However, the systematic or large-scale analysis of this PTM is hampered by several factors, including low stoichiometry and the lability of the O-glycosidic bond during tandem mass spectrometry. Using a library of 72 synthetic glycopeptides, we developed a two-stage tandem MS approach consisting of pulsed Q dissociation (PQD) for O-GlcNAc peptide detection and electron transfer dissociation (ETD) for identification and site localization. Based on a set of O-GlcNAc specific fragment ions, we further developed a score (OScore) that discriminates O-GlcNAc peptide spectra from spectra of unmodified peptides with 95% sensitivity and 999% specificity. Integrating the OScore into the two-stage LC-MS/MS approach detected O-GlcNAc peptides in the low fmol range and at 10-fold better sensitivity than a single data-dependent ETD tandem MS experiment.
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