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) for peroxide reduction and a resolving cysteine (C) for C regeneration. C can be readily sulfonated to C-SOH by protracted oxidative stress, which inactivates Prx1 as a peroxidase. By comparison, sulfonation of C to C-SOH in mammalian cells has only been reported once. The rare report of C sulfonation prompts the following questions: "can C-SOH be detected more readily with the current high sensitivity mass spectrometers (MS)?" and "do C and C have distinct propensities to sulfonation?" Answers to these questions could shed light on how differential sulfonation of C and C regulates Prx1 functions in cells. We used a sensitive Orbitrap MS to analyze both basal and HO-induced sulfonation of C and C 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 HO, both C and C were partially sulfonated in either rPrx1 or cPrx1. Still, exogenous HO heightened the sulfonation levels of both C and C by ~200-700%. Titration with HO revealed that C and C possessed distinct propensities to sulfonation. This surprising discovery of prevalent Prx1 C sulfonation affords a motivation for future investigation of its precise functions in cellular stress response.