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

摘要: 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 high performance liquid chromatography (HPLC) method quantifying the amount Prx2 in red blood cells (RBCs). RBC proteins were separated using HPLC, and single peak was detected that matched produced recombinant Prx2. Under improved conditions, calibration curve (reduced form) linear over range 0.5-20.0 μg with correlation coefficient 0.999. The minimum detectable level 0.2 μg, signal-to-noise ratio 3 per 20 μl injection volume. SDS-PAGE mass spectrometric analysis showed comprising almost exclusively Further high-resolution nanoLC–MS/MS demonstrated oxidation sensitive, Cys-51 carbamidomethylated iodoacetamide-alkylation during in-gel digestion but not modified sulfinic acid (−SO2H) or sulfonic (−SO3H). These results indicated reduced form hyperoxidized form. basic experiments allowed us determine relative amounts native RBCs taken from healthy subjects. average levels male female subjects 7.28 ng/mg 8.29 ng/mg, respectively, no significant difference observed between sexes. Therefore, HPLC UV detection described herein offers convenient quantitatively its decrease human RBCs.