Mass Spectrometry-based Characterization of Protein Glutathionylspermidine Modification Using Complementary Dissociation Approaches

摘要

Protein S-thiolation with small thiol containing molecules, including glutathione and cysteine, was found in many physiological circumstances and accorded an important role for oxidative defense. However, this process is not completely understood due to lack of appropriate methods to enrich S-thiolated proteins. A number of glutathionylspermidine (Gsp) conjugates from pathogenic microbes have been identified as potent biological antioxidants for protein. Treatment of E. coli with H_(2)O_(2) would result in transient Gsp accumulation through selective inhibition of Gsp amidase activity, indicating that Gsp could be a critical component against oxidative stress. Due to the distinct systems for thiol balance in host and pathogen, investigation of the metabolism for GSH-spermidine conjugate is highly relevant for anti-parasite therapy. Herein, using the synthetic tryptic peptides derived from cysteine-containing active site of a protein tyrosine phosphatase and the transcription factor OxyR as two models, we investigated and identified the MS/MS fragmentation patterns specifically associated with protein Gspmodification, under collision-induced dissociation (CID), electron transfer dissociation (ETD) and higher-energy C-trap dissociation (HCD). ETD was performed on LTQ XL ETD, HCD was on LTQ Orbitrap XL hybrid mass spectrometry. Additionally, 4700 MALDI-TOF/TOF was also applied to glutathionylated and Gsp-modified peptides for high energy CID. On the other hand, the intact masses of modified and unmodified proteins were determined by ESI-MS on the QSTAR-XL hybrid quadrupole time-of-flight mass spectrometer with a home-made nanosprayer applied with -3.5 kV.