Thiol‐blocking electrophiles interfere with labeling and detection of protein sulfenic acids

Abstract

Cellular exposure to reactive oxygen species induces the rapid oxidation of DNA, proteins, lipids, and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is a chemical blocking of free thiols with reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide, and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are susceptible to reduction by DTT, TCEP, and ascorbate, regenerating protein thiols, or in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.

DYn-2 Based Identification of Arabidopsis Sulfenomes*

“…It is noteworthy to mention that the use of alkylating agents such as IAM and MMTS is not recommended, as they show reactivity with DYn-2 (unpublished data). Moreover, IAM, NEM, and MMTS are also known to form adducts with Cys-SOH, cleavable under reducing conditions (20). Harvested cells were ground on ice using sand with extraction buffer (25 mM Tris-HCl pH 7.6, 15 mM MgCl 2 , 150 mM NaCl, 15 mM pNO 2 PhenylPO 4 , 60 mM B-glycerolphosphate, 0.1% Nonidet P-40, 0.1 mM Na 3 VO 4 , 1 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 1 M E64, 1ϫ Roche protease inhibitor mixture, 5% ethylene glycol) supplemented with catalase (bovine liver, Sigma-Aldrich, St Louis, MO) at 200 U/ml.…”

The Expanding Landscape of the Thiol Redox Proteome

“…The principal issue is that the thiol-blocking agents commonly used in BST methods display reactivity with cysteine S-sulfenic acid, which is a key intermediate in thiol oxidation (54,55). Uncertainty regarding the species labeled by thiol-blocking agents introduces ambiguity in interpretation.…”

ProteinS-Glutathionylation Mediates Macrophage Responses to Metabolic Cues from the Extracellular Environment

“…NEM was added prelysis to avoid thiol oxidation during cell lysis and other processing steps and to avoid loss of protein thiol-GSH mixed disulfides due to transthiolation and disulfide formation during sample preparation (18). NEM is a commonly used thiol-alkylating agent because of its high reactivity and specificity for thiols at physiological pH (6.5-7.5); at higher pH (>7.5), NEM reacts with lysines, arginines, histidines, and tyrosines (37,46) and has been reported to also react with sulfenic acids (49) and possibly with sulfinic acids (29,49). NEM has proven to be a better overall alkylating agent than IAM based on its ability to react with free thiols and sulfenic acids faster, more efficiently, and within a broader effective pH range (49,51).…”

“…NEM is a commonly used thiol-alkylating agent because of its high reactivity and specificity for thiols at physiological pH (6.5-7.5); at higher pH (>7.5), NEM reacts with lysines, arginines, histidines, and tyrosines (37,46) and has been reported to also react with sulfenic acids (49) and possibly with sulfinic acids (29,49). NEM has proven to be a better overall alkylating agent than IAM based on its ability to react with free thiols and sulfenic acids faster, more efficiently, and within a broader effective pH range (49,51). Sulfonic acids (-SO 3 H) and S-nitrosylated thiols (-SNO) appear to be generally unreactive with thiolalkylating reagents, including NEM and IAM (29,49).…”

“…NEM has proven to be a better overall alkylating agent than IAM based on its ability to react with free thiols and sulfenic acids faster, more efficiently, and within a broader effective pH range (49,51). Sulfonic acids (-SO 3 H) and S-nitrosylated thiols (-SNO) appear to be generally unreactive with thiolalkylating reagents, including NEM and IAM (29,49). Cells were then lysed using a mild lysis buffer (10 mM NEM, 50 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, and 0.1% NP-40 with protease inhibitor cocktail [Roche], pH 7.4), and lysates were sonicated and centrifuged to remove cellular debris.…”