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Review
. 2010:473:179-97.
doi: 10.1016/S0076-6879(10)73009-3.

Measurement and identification of S-glutathiolated proteins

Bradford G Hill  1 , Kota V Ramana, Jian Cai, Aruni Bhatnagar, Satish K Srivastava
Affiliations
Review

Measurement and identification of S-glutathiolated proteins

Bradford G Hill et al. Methods Enzymol. 2010.

Abstract

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.

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Figures

Figure 1
Figure 1
Analysis of reduced and glutathiolated actin by mass spectrometry. (A) Deconvoluted ESI+/MS spectra of actin before (upper panel) and after (lower panel) modification by GSNO. The protein was first reduced by DTT and excess DTT was removed by Sephadex gel filtration. The reduced protein was then added to acetonitrile:water:acetic acid for spectrometric analysis (upper panel). The reduced protein was then incubated with 1 mM GSNO (in 20 mM Tris, pH 7.5) for 1 h. The protein was desalted and then analyzed by ESI/MS(lower panel). Note the +305 Da shift in the mass of the major ion, indicating the adduction of a single glutathione molecule to one molecule of actin. (B)ESI/MS spectra of native and GS-actin after hydrolysis with Glu-C. Two peaks (m/z 2640 and 3820) were observed only in the GS-actin spectrum and those corresponding to the addition of 305 Da to peaks from the sample of the native protein (m/z 2335 and 3515) were identified.
Figure 2
Figure 2
Immunological detection of glutathiolated proteins. (A) Photomicrographs of aortic rings stained with the anti-glutathione antibody. Aortic rings were dissected from adult male rats, mounted ex vivo in a perfusion bath, and pre-contracted with 1 μM phenylephrine. The rings were either left untreated (control), stimulated with 1 μM acetylcholine (+Ach), or treated with acetylcholine in the presence of 100 μM L-NAME, a NO synthase inhibitor. Immediately after treatment, the rings were fixed and stained with the anti-glutathione antibody (1:200 dilution). (B) Two-dimensional Western blots of aortic rings precontracted with phenylephrine and relaxed by acetylcholine in the absence or presence of L-NAME. Extracts of aortic rings were subjected to 2D Western blot analysis using the anti-glutathione antibody. Note: The major immunopositive spot corresponds to actin (as indicated in the figure). (C) One-dimensional SDS-PAGE of rabbit skeletal muscle actin before (−) and after (+) treatment with 1 mM GSNO. The treated and untreated proteins were separated by SDS-PAGE, and Western blots were developed with the anti-glutathione (anti-PSSG) and anti-actin antibodies.
Scheme 1
Scheme 1
Mechanisms of protein S-glutathiolation by nitric oxide and reactive oxygen species. In most cells, GSSG and nitrosoglutathione (GSNO) are likely to be the most significant glutathiolating agents. The cellular abundance of GSSG is regulated by processes that generate GSSG, such as the reduction of peroxides by glutathione peroxidases (GP). GSNO formed after direct reaction of NO with thiyl radicals or after reaction of glutathione with advanced nitrogen oxide species (e.g., N2O3) enters into transnitrosation reactions that also result in S-glutathiolated proteins. Peroxynitrite (ONOO−), formed from the reaction of NO with superoxide, is able to mediate the formation of both S-nitrosated (PS-NO) and sulfenic acid-modified (PS-OH) proteins and glutathione; these “activated” thiols react readily to form protein-glutathione adducts. Hydrogen peroxide also promotes PS-OH/GS-OH formation that leads to protein glutathiolation. Protein glutathiolation can facilitate redox cell signaling, regulate enzyme function, protect protein thiols from advanced protein oxidation, and, in some cases, promote cell death.
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