title: A simple high performance liquid chromatography method for quantitatively determining the reduced form of peroxiredoxin 2 and the mass spectrometric analysis of its oxidative status.
abstract: Peroxiredoxins (Prxs) are a family of thiol peroxidases, which have been suggested to serve as biomarkers for diseases caused by oxidative stress. In this study, we established a high performance liquid chromatography (HPLC) method for quantifying the amount of Prx2 in red blood cells (RBCs). RBC proteins were separated using HPLC, and a single peak was detected that matched that produced by recombinant Prx2. Under improved conditions, the calibration curve for Prx2 (reduced form) was linear over the range of 0.5-20.0μg with a correlation coefficient of 0.999. The minimum detectable level of the recombinant Prx2 was 0.2μg, with a signal-to-noise ratio of 3 per 20μl of injection volume. SDS-PAGE and mass spectrometric analysis showed that the proteins comprising the peak were almost exclusively Prx2. Further high-resolution analysis using nanoLC-MS/MS demonstrated that the oxidation sensitive, Cys-51 was carbamidomethylated by iodoacetamide-alkylation during in-gel digestion but was not modified with sulfinic acid (-SO2H) or sulfonic acid (-SO3H). These results indicated that the separated Prx2 was the reduced form and not the hyperoxidized form. These basic experiments allowed us to determine the relative amounts of native Prx2 in RBCs taken from healthy subjects. The average levels of Prx2 in male and female subjects were 7.28ng/mg and 8.29ng/mg, respectively, and no significant difference was observed between the sexes. Therefore, the HPLC method with UV detection described herein offers a convenient method to quantitatively determine the levels of reduced form of Prx2 and its oxidative decrease in human RBCs.
authors.affiliation: Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. Electronic address: yo@my-pharm.ac.jp.
authors.forename: Y
authors.initials: Y
authors.lastname: Ogasawara
authors.affiliation: Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: Yi
authors.initials: Y
authors.lastname: Ishida
authors.affiliation: Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Takikawa
authors.affiliation: Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Funaki
authors.affiliation: Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Koike
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000097:blood* / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D054464:Peroxiredoxins
keywords: Biomarker; High performance liquid chromatography; Oxidative stress; Peroxiredoxin 2; Red blood cell