Please use this identifier to cite or link to this item: https://doi.org/10.1021/ac401821d
Title: Multiple reaction monitoring mass spectrometry for the discovery and quantification of o-glcnac-modified proteins
Authors: Maury, J.J.P.
Ng, D.
Bi, X.
Bardor, M.
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

Show full item record
Files in This Item:
There are no files associated with this item.

SCOPUSTM   
Citations

18
checked on Apr 5, 2020

WEB OF SCIENCETM
Citations

21
checked on Mar 20, 2020

Page view(s)

87
checked on Mar 29, 2020

Google ScholarTM

Check

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.