Artificial Oxidation of Cysteine Residues in Peroxiredoxin 6 Detected by Twodimensional Gel Electrophoresis and Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquidchromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). Weused Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidationof the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid atthe 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot ofPrx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6),and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidizedPrx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from thebasic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes afterisoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological orartificial, when an oxidized cysteine residue(s) is identified.