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

Abstract 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.