Characteristic tandem mass spectral features under various collision chemistries for site-specific identification of protein S-glutathionylation.
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.
Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, 11529, Taiwan, sandra77@gate.sinica.edu.tw.
Chi-Chi
CC
Chou
Bing-Yu
BY
Chiang
Jason Ching-Yao
JC
Lin
Kuan-Ting
KT
Pan
Chun-Hung
CH
Lin
Kay-Hooi
KH
Khoo
D000595:Amino Acid Sequence; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017293:Protein S / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010446:Peptide Fragments; D017293:Protein S; D005978:Glutathione