Measurement and identification of S-glutathiolated proteins.
Protein thiol modifications occur under both physiological and pathological conditions and can regulate protein function, redox signaling, and cell viability. The thiolation of proteins by glutathione (GSH) appears to be a particularly important mode of posttranslational modification that is increased under conditions of oxidative or nitrosative stress. Modification of proteins by glutathiolation has been shown to affect the structure and function of several susceptible proteins and protect them from subsequent oxidative injury. In many cases, the glutathiolated proteins are low in abundance, and dethiolation occurs readily. Therefore, sensitive, reliable, and reproducible methods are required for measuring both the total levels of protein glutathiolation and for identifying glutathiolated proteins under given conditions. These methods necessitate the preservation or the controlled removal of the GSH adducts during sample preparation for the accurate measurement of total S-glutathiolation and for the identification of protein-GSH adducts. In this chapter, we briefly review and provide protocols for chemical, mass spectrometric, immunological, and radioactive tagging techniques, for measuring protein S-glutathiolation in cells and tissues.
Diabetes and Obesity Center, University of Louisville, Louisville, Kentucky, USA.
Bradford G
BG
Hill
Kota V
KV
Ramana
Jian
J
Cai
Aruni
A
Bhatnagar
Satish K
SK
Srivastava
D000818:Animals; D002477:Cells / Q000737:chemistry; D019411:Clinical Laboratory Techniques; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000378:metabolism; D013451:Sulfonic Acids / Q000032:analysis / Q000378:metabolism; D014020:Tissue Extracts / Q000737:chemistry
D011506:Proteins; D013438:Sulfhydryl Compounds; D013451:Sulfonic Acids; D014020:Tissue Extracts; D005978:Glutathione