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|Title:||Multiple reaction monitoring mass spectrometry for the discovery and quantification of o-glcnac-modified proteins||Authors:||Maury, J.J.P.
CHOO BOON HWA,ANDRE
|Issue Date:||7-Jan-2014||Citation:||Maury, J.J.P., Ng, D., Bi, X., Bardor, M., CHOO BOON HWA,ANDRE (2014-01-07). Multiple reaction monitoring mass spectrometry for the discovery and quantification of o-glcnac-modified proteins. Analytical Chemistry 86 (1) : 395-402. ScholarBank@NUS Repository. https://doi.org/10.1021/ac401821d||Abstract:||O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification regulating proteins involved in a variety of cellular processes and diseases. Unfortunately, O-GlcNAc remains challenging to detect and quantify by shotgun mass spectrometry (MS) where it is time-consuming and tedious. Here, we investigate the potential of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted MS method, to detect and quantify native O-GlcNAc modified peptides without extensive labeling and enrichment. We report the ability of MRM-MS to detect a standard O-GlcNAcylated peptide and show that the method is robust to quantify the amount of O-GlcNAcylated peptide with a method detection limit of 3 fmol. In addition, when diluted by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated peptide remains detectable. Next, we apply this strategy to study glycogen synthase kinase-3 beta (GSK-3β), a kinase able to compete with O-GlcNAc transferase and modify identical site on proteins. We demonstrate that GSK-3β is itself modified by O-GlcNAc in human embryonic stem cells (hESC). Indeed, by only using gel electrophoresis to grossly enrich GSK-3β from whole cell lysate, we discover by MRM-MS a novel O-GlcNAcylated GSK-3β peptide, bearing 3 potential O-GlcNAcylation sites. We confirm our finding by quantifying the increase of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase inhibitor. This novel O-GlcNAcylation could potentially be involved in an autoinhibition mechanism. To the best of our knowledge, this is the first report utilizing MRM-MS to detect native O-GlcNAc modified peptides. This could potentially facilitate rapid discovery and quantification of new O-GlcNAcylated peptides/proteins. © 2013 American Chemical Society.||Source Title:||Analytical Chemistry||URI:||http://scholarbank.nus.edu.sg/handle/10635/87954||ISSN:||00032700||DOI:||10.1021/ac401821d|
|Appears in Collections:||Staff Publications|
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