TY - JOUR
AU - Khoo, Kay-Hooi
AB - Protein S-glutathionylation is a reversible post-translational modification widely implicated in redox regulated biological functions. Conventional biochemical methods, however, often do not allow such a mixed disulfide modification to be reliably identified on specific cysteine residues or be distinguished from other related oxidized forms. To develop more efficient mass spectrometry (MS)-based analytical strategies for this purpose, we first investigated the MS/MS fragmentation pattern of S-glutathionylated peptides under various dissociation modes, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD), using synthetic peptides derived from protein tyrosine phosphatase as models. Our results indicate that a MALDI-based high energy CID MS/MS on a TOF/TOF affords the most distinctive spectral features that would facilitate rapid and unambiguous identification of site-specific S-glutathionylation. For more complex proteomic samples best tackled by LC-MS/MS approach, we demonstrate that HCD performed on an LTQ-Orbitrap hybrid instrument fairs better than trap-based CID and ETD in allowing more protein site-specific S-glutathionylation to be confidently identified by direct database searching of the generated MS/MS dataset using Mascot. Overall, HCD afforded more peptide sequence-informative fragment ions retaining the glutathionyl modification with less neutral losses of side chains to compromise scoring. In conjunction with our recently developed chemo-enzymatic tagging strategy, our nanoLC-HCD-MS/MS approach is sufficiently sensitive to identify endogenous S-glutathionylated peptides prepared from non-stressed cells. It is anticipated that future applications to global scale analysis of protein S-glutathionylation will benefit further from current advances in both speed and mass accuracy afforded by HCD MS/MS mode on the Orbitrap series.
TI - Characteristic Tandem Mass Spectral Features Under Various Collision Chemistries for Site-Specific Identification of Protein S-Glutathionylation
JF - Journal of the American Society for Mass Spectrometry
DO - 10.1007/s13361-014-1014-9
DA - 2014-11-06
UR - https://www.deepdyve.com/lp/springer-journals/characteristic-tandem-mass-spectral-features-under-various-collision-d5rkDdztW2
SP - 120
EP - 132
VL - 26
IS - 1
DP - DeepDyve
ER -