Sulfonation of the resolving cysteine in human peroxiredoxin 1: A comprehensive analysis by mass spectrometry

Peroxiredoxin 1 (Prx1) is an essential peroxidase that reduces cellular peroxides. It holds 2 indispensable cysteines for its activity: a peroxidatic cysteine (C_P) for peroxide reduction and a resolving cysteine (C_R) for C_P regeneration. C_P can be readily sulfonated to C_P-SO₃H by protracted oxidative stress, which inactivates Prx1 as a peroxidase. By comparison, sulfonation of C_R to C_R-SO₃H in mammalian cells has only been reported once. The rare report of C_R sulfonation prompts the following questions: "can C_R-SO₃H be detected more readily with the current high sensitivity mass spectrometers (MS)?" and "do C_P and C_R have distinct propensities to sulfonation?" Answers to these questions could shed light on how differential sulfonation of C_P and C_R regulates Prx1 functions in cells. We used a sensitive Orbitrap MS to analyze both basal and H₂O₂-induced sulfonation of C_R and C_P in either recombinant human Prx1 (rPrx1) or HeLa cell Prx1 (cPrx1). In the Orbitrap MS, we optimized both collision-induced dissociation and higher-energy collisional dissociation methods to improve the analytical sensitivity of cysteine sulfonation. In the basal states without added H₂O₂, both C_P and C_R were partially sulfonated in either rPrx1 or cPrx1. Still, exogenous H₂O₂ heightened the sulfonation levels of both C_P and C_R by ~200-700%. Titration with H₂O₂ revealed that C_P and C_R possessed distinct propensities to sulfonation. This surprising discovery of prevalent Prx1 C_R sulfonation affords a motivation for future investigation of its precise functions in cellular stress response.