1 research outputs found
Multiple Reaction Monitoring Mass Spectrometry for the Discovery and Quantification of O‑GlcNAc-Modified Proteins
O-linked <i>N</i>-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