Profiling Protein S-Sulfination with Maleimide-Linked Probes

Abstract

Cysteine residues are susceptible to oxidation to form S-sulfinyl (R-SO₂ H) and S-sulfonyl (R-SO₃ H) post-translational modifications. Here we present a simple bioconjugation strategy to label S-sulfinated proteins by using reporter-linked maleimides. After alkylation of free thiols with iodoacetamide, S-sulfinated cysteines react with maleimide to form a sulfone Michael adduct that remains stable under acidic conditions. Using this sequential alkylation strategy, we demonstrate differential S-sulfination across mouse tissue homogenates, as well as enhanced S-sulfination following pharmacological induction of endoplasmic reticulum stress, lipopolysaccharide stimulation, and inhibitors of the electron transport chain. Overall, this study reveals a broadened profile of maleimide reactivity across cysteine modifications, and outlines a simple method for profiling the physiological role of cysteine S-sulfination in disease.

Keywords: Michael addition; cysteine; protein modifications; redox chemistry; sulfur.