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Title: Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling

Abstract

Protein S-glutathionylation (SSG) is an important regulatory posttranslational modification of protein cysteine (Cys) thiol redox switches, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols and enrichment using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys-sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG-modification compared to controls.. This approach was extended to identify potential SSG modified Cys-sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys-sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment. These proteins covered a range of molecular types and molecular functions with freemore » radical scavenging, and cell death and survival included as the most significantly enriched functional categories. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of S-glutathionylated proteins. The analytical strategy also provides a unique approach to determining the major pathways and cell processes most susceptible to glutathionylation at a proteome-wide scale.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1130232
Report Number(s):
PNNL-SA-96702
40991; 400412000
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Free Radical Biology & Medicine, 67:460-470
Additional Journal Information:
Journal Name: Free Radical Biology & Medicine, 67:460-470
Country of Publication:
United States
Language:
English
Subject:
Environmental Molecular Sciences Laboratory

Citation Formats

Su, Dian, Gaffrey, Matthew J., Guo, Jia, Hatchell, Kayla E., Chu, Rosalie K., Clauss, Therese RW, Aldrich, Joshua T., Wu, Si, Purvine, Samuel O., Camp, David G., Smith, Richard D., Thrall, Brian D., and Qian, Weijun. Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling. United States: N. p., 2014. Web. doi:10.1016/j.freeradbiomed.2013.12.004.
Su, Dian, Gaffrey, Matthew J., Guo, Jia, Hatchell, Kayla E., Chu, Rosalie K., Clauss, Therese RW, Aldrich, Joshua T., Wu, Si, Purvine, Samuel O., Camp, David G., Smith, Richard D., Thrall, Brian D., & Qian, Weijun. Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling. United States. doi:10.1016/j.freeradbiomed.2013.12.004.
Su, Dian, Gaffrey, Matthew J., Guo, Jia, Hatchell, Kayla E., Chu, Rosalie K., Clauss, Therese RW, Aldrich, Joshua T., Wu, Si, Purvine, Samuel O., Camp, David G., Smith, Richard D., Thrall, Brian D., and Qian, Weijun. Tue . "Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling". United States. doi:10.1016/j.freeradbiomed.2013.12.004.
@article{osti_1130232,
title = {Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling},
author = {Su, Dian and Gaffrey, Matthew J. and Guo, Jia and Hatchell, Kayla E. and Chu, Rosalie K. and Clauss, Therese RW and Aldrich, Joshua T. and Wu, Si and Purvine, Samuel O. and Camp, David G. and Smith, Richard D. and Thrall, Brian D. and Qian, Weijun},
abstractNote = {Protein S-glutathionylation (SSG) is an important regulatory posttranslational modification of protein cysteine (Cys) thiol redox switches, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols and enrichment using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys-sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG-modification compared to controls.. This approach was extended to identify potential SSG modified Cys-sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys-sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment. These proteins covered a range of molecular types and molecular functions with free radical scavenging, and cell death and survival included as the most significantly enriched functional categories. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of S-glutathionylated proteins. The analytical strategy also provides a unique approach to determining the major pathways and cell processes most susceptible to glutathionylation at a proteome-wide scale.},
doi = {10.1016/j.freeradbiomed.2013.12.004},
journal = {Free Radical Biology & Medicine, 67:460-470},
number = ,
volume = ,
place = {United States},
year = {2014},
month = {2}
}