Abstract
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.
Highlights
During the past decade, proteome analysis has utilized two-dimensional gel electrophoresis (2D-GE) for protein separation and peptide mass finger printing using mass spectrometry.1 In an extensive research on proteome analysis, protein identification and post-translational modifications have been major research fields
We prepared two types of oxidized peroxiredoxin 6 (Prx6): carrying oxidized Cys-47, and other carrying both oxidized Cys-47 and Cys-91. Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE
Upon H2O2 treatment, the basic spot of Prx6 disappeared and an acidic spot of Prx6 appeared at 28.7 kDa and a pI of 6.2 (Figure 1B)
Summary
Proteome analysis has utilized two-dimensional gel electrophoresis (2D-GE) for protein separation and peptide mass finger printing using mass spectrometry. In an extensive research on proteome analysis, protein identification and post-translational modifications have been major research fields. Proteome analysis has utilized two-dimensional gel electrophoresis (2D-GE) for protein separation and peptide mass finger printing using mass spectrometry.. In an extensive research on proteome analysis, protein identification and post-translational modifications have been major research fields. Protein modifications caused by oxidative stress, such as oxidation of amino acid side chains, are important therapeutic targets.. Cysteine is an amino acid residue sensitive to oxidative reaction. The sulfhydryl group of cysteine residue exists as free-SH and -S-S-, and in an overoxidation form in vivo and in vitro.. Overoxidation products of cysteine residue are cysteine sulfinic acid (-SO2H) and cysteine sulfonic acid (-SO3H), which are yielded by an oxidation reaction with peroxide.. Overoxidation products of cysteine residue are cysteine sulfinic acid (-SO2H) and cysteine sulfonic acid (-SO3H), which are yielded by an oxidation reaction with peroxide.7 The sulfhydryl group of cysteine residue exists as free-SH and -S-S-, and in an overoxidation form in vivo and in vitro. Overoxidation products of cysteine residue are cysteine sulfinic acid (-SO2H) and cysteine sulfonic acid (-SO3H), which are yielded by an oxidation reaction with peroxide. Oxidation of this residue
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