Mass Spectrometry Letters

Korean Society for Mass Spectrometry (한국질량분석학회)
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Artificial Oxidation of Cysteine Residues in Peroxiredoxin 6 Detected by Twodimensional Gel Electrophoresis and Capillary Liquid Chromatography-Electrospray Mass Spectrometry

  • Kimata, Junko (Thermo Fisher Scientific) ;
  • Shigeri, Yasushi (Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)) ;
  • Yoshida, Yasukazu (Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)) ;
  • Niki, Etsuo (Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)) ;
  • Kinumi, Tomoya (National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (AIST))
  • Received : 2011.12.12
  • Accepted : 2012.02.21
  • Published : 2012.03.15
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Abstract

Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 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 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. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.

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