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. 2017 Jul:108:785-792.
doi: 10.1016/j.freeradbiomed.2017.04.341. Epub 2017 Apr 25.

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

Changgong Wu  1 , Huacheng Dai  1 , Lin Yan  1 , Tong Liu  1 , Chuanglong Cui  1 , Tong Chen  1 , Hong Li  2
Affiliations

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

Changgong Wu et al. Free Radic Biol Med. 2017 Jul.

Abstract

Peroxiredoxin 1 (Prx1) is an essential peroxidase that reduces cellular peroxides. It holds 2 indispensable cysteines for its activity: a peroxidatic cysteine (CP) for peroxide reduction and a resolving cysteine (CR) for CP regeneration. CP can be readily sulfonated to CP-SO3H by protracted oxidative stress, which inactivates Prx1 as a peroxidase. By comparison, sulfonation of CR to CR-SO3H in mammalian cells has only been reported once. The rare report of CR sulfonation prompts the following questions: "can CR-SO3H be detected more readily with the current high sensitivity mass spectrometers (MS)?" and "do CP and CR have distinct propensities to sulfonation?" Answers to these questions could shed light on how differential sulfonation of CP and CR regulates Prx1 functions in cells. We used a sensitive Orbitrap MS to analyze both basal and H2O2-induced sulfonation of CR and CP 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 CP and CR were partially sulfonated in either rPrx1 or cPrx1. Still, exogenous H2O2 heightened the sulfonation levels of both CP and CR by ~200-700%. Titration with H2O2 revealed that CP and CR possessed distinct propensities to sulfonation. This surprising discovery of prevalent Prx1 CR sulfonation affords a motivation for future investigation of its precise functions in cellular stress response.

Keywords: Mass spectrometry; Oxidative stress; Peroxiredoxin; Sulfonation.

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Conflict of interest statement

Disclosures

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. SDS-PAGE and Western blotting of sulfonated rPrx1 following H2O2 treatment
rPrx1 was reduced with 100 mM DTT for 1 h at 37 °C. After acetone precipitation to remove the DTT, reduced rPrx1 were treated without or with 1 mM of H2O2. The resulting proteins were separated using either non-reducing (A) or reducing (B) 12% SDS PAGE gels. Two hundred ng of rPrx1 in each sample was analyzed by Western blotting with an anti-Prx-SO3 antibody.
Fig. 2
Fig. 2. CID spectra of Prx1 (amino acids 38–62) with CP-SO3H or CP alkylation
(A) An MS/MS spectrum of a 3H+ ion at m/z 1028.83 for the sulfonated peptide. (B) An MS/MS spectrum of a 3H+ ion at m/z 1031.84 the alkylated peptide. Sulfonated rPrx1 was obtained from the reducing gels outlined in Fig. 1. The strings of b- and y-series ions from the MS/MS spectra matched to 38-VVFFFYPLDFTFVCPTEIIAFSDR-62 in human Prx1. The sulfonated peptide with a Cys +47.98 amu was identified with a Mascot score of 51 (A). The alkylated peptide with a Cys +57.02 amu was identified with a Mascot score of 70 (B). The PTMs were found between b14 and b15 and between y11 and y12 ions in each spectrum (marked red), which confirmed the sulfonation and alkylation of CP in rPrx 1.
Fig. 3
Fig. 3. CID spectra of Prx1 (amino acids 169–190) with CR-SO3H or CR alkylation
(A) An MS/MS spectrum of a 3H+ ion at m/z 799.73 for the sulfonated peptide. (B) An MS/MS spectrum of a 3H+ ion at m/z 802.74 the alkylated peptide. Sulfonated rPrx1 was obtained from the reducing gels outlined in Fig. 1. The strings of b- and y-series ions from the MS/MS spectra matched to 169-HGEVCPAGWKPGSDTIKPDVQK-190 in human Prx1. The sulfonated peptide with a Cys +47.98 amu was identified with a Mascot score of 52 (A). The alkylated peptide with a Cys +57.02 amu was identified with a Mascot score of 68 (B). The PTMs were found between b4 and b5 and between y17 and y18 ions in each spectrum (marked red), which confirmed the sulfonation and alkylation of CR in rPrx 1.
Fig. 4
Fig. 4. Comparison of H2O2-induction of CP–SO3H and CR–SO3H in rPrx1
Following a 30-min and 1 mM H2O2 treatment, sulfonated rPrx1 was obtained from the reducing gels outlined in Fig. 1 and analyzed by LC/MS/MS as described in the Materials and Methods. Extracted ion chromatograms (XIC) for the precursor ions that matched to the peptides containing either CP-SO3H or CR-SO3H were obtained by Skyline. The XIC signals for the sulfonated peptides were normalized to the combined XIC of all the identified rPrx1peptide ions. Sulfonation change is presented here as the fold change (%) over a control with no added H2O2 oxidation, whose value is set as 100%. The changes were considered statistically significant based on the Student’s T-test (N=3/group).
Fig. 5
Fig. 5. Western blots for Prx1-SO3 in HeLa cells
Cells were treated for 30 min with either media alone or supplemented with 1 mM of H2O2. Sixty µg of proteins from each sample were separated using a 12% reducing SDS-PAGE gel. (A) An anti-Prx1-SO3 antibody (Abcam, 1:2500 dilution) was used to detect Prx1-SO3. Total Prx1 was detected using an anti-Prx1 antibody. (B) Substantial basal Prx1-SO3 was readily observed in the untreated cells and significantly increased Prx1-SO3 was detected after the H2O2 treatment (N=3/group).
Fig. 6
Fig. 6. Comparison of H2O2-induction of CP–SO3H and CR–SO3H in cPrx1
Following a 30-min and 1 mM H2O2 treatment of the HeLa cells, peptide sulfonation events were analyzed and quantified as described in Fig. 4 (N=3/group).
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