Protein carbonyl detection by two-dimensional fluorescence difference gel electrophoresis

DOI 被引用文献1件 参考文献46件 オープンアクセス
  • Sawada Akira
    Department of Physics, Kitasato University School of Science
  • Ueno Takeshi
    Department of Pharmacology and Toxicology, Kyorin University School of Medicine
  • Kawashima Yusuke
    Department of Physics, Kitasato University School of Science
  • Haruta-Satoh Eri
    Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University
  • Oh-Ishi Masamichi
    Department of Physics, Kitasato University School of Science
  • Kodera Yoshio
    Department of Physics, Kitasato University School of Science
  • Maeda Tadakazu
    Department of Physics, Kitasato University School of Science

書誌事項

公開日
2008
DOI
  • 10.2198/jelectroph.52.9
公開者
日本電気泳動学会

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説明

Oxidative stress is implicated in a broad variety of chronic and acute diseases, including Alzheimer's disease, arteriosclerosis and diabetes. In order to study oxidative stress at the proteome level proteins were labeled with Cy2NHS (the N-hydroxyl succinimidyl ester derivative of a cyanine dye reactive with the ε-amino group of lysine residues), or with Cy3- and Cy5HZ (hydrazide derivatives of the dyes reactive with protein carbonyls) and analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Two samples were used for testing the CyHZ labeling; one is low molecular mass marker proteins artificially oxidized by NaOCl and the other renal cortex extracts of 30-week-old diabetes model OLETF (Otsuka Long-Evans Tokushima Fatty) and control LETO (Long-Evans Tokushima Otsuka) rats. Spot volumes Vni and contrasts cni of 2D-DIGE images were quantified following background subtraction, where the suffices n (=2, 3 or 5) and i (=1, 2, ...) represent the Cy-signal and the Cy2-detected spot identification numbers, respectively. The contrasts c3i and c 5i were on average about 0.53 times as high as c2i. Taking the contrast into account, a simple parameter qni was proposed for judging the quality of spot images. The difference in-gel analysis (DIA) algorithm should be used in combination with the quality parameters to identify statistical outliers as candidates for abnormally carbonylated proteins. Inspection of the background flatness around the candidate spots is also required before making a conjecture as to whether the candidate protein is abnormally oxidized.<br>

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