title: Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling.
abstract: S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, 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 covalent capture of reduced thiols 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 initially 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, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible to S-glutathionylation under stress conditions.
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Dian
authors: D
authors: Su
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Matthew J
authors: MJ
authors: Gaffrey
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Jia
authors: J
authors: Guo
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Kayla E
authors: KE
authors: Hatchell
authors: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Rosalie K
authors: RK
authors: Chu
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Therese R W
authors: TR
authors: Clauss
authors: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Joshua T
authors: JT
authors: Aldrich
authors: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Si
authors: S
authors: Wu
authors: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Sam
authors: S
authors: Purvine
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: David G
authors: DG
authors: Camp
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Richard D
authors: RD
authors: Smith
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: Brian.Thrall@pnnl.gov.
authors: Brian D
authors: BD
authors: Thrall
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: Weijun.qian@pnnl.gov.
authors: Wei-Jun
authors: WJ
authors: Qian
mesh_terms: D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D013194:Staining and Labeling
chemical_list: D020543:Proteome; D003958:Diamide; D006861:Hydrogen Peroxide; D005978:Glutathione; D003545:Cysteine
keywords: Hydrogen peroxide; Macrophage; Protein thiols; Proteomics; Redox regulation; Resin-assisted enrichment; S-Glutathionylation