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)-SO(3)H by protracted oxidative stress, which inactivates Prx1 as a peroxidase. By comparison, sulfonation of C(R) to C(R)-SO(3)H in mammalian cells has only been reported once. The rare report of C(R) sulfonation prompts the following questions: "can C(R)-SO(3)H 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 H(2)O(2)-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 H(2)O(2), both C(P) and C(R) were partially sulfonated in either rPrx1 or cPrx1. Still, exogenous H(2)O(2) heightened the sulfonation levels of both C(P) and C(R) by ~200-700%. Titration with H(2)O(2) 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.