期刊
INTERNATIONAL JOURNAL OF MASS SPECTROMETRY
卷 305, 期 2-3, 页码 217-227出版社
ELSEVIER
DOI: 10.1016/j.ijms.2010.11.010
关键词
High resolution proteomics; Myofibrillar muscle protein; FTICR mass spectrometry; Post-mortem degradation; Oxidative modification; 2D-gel electrophoresis
资金
- Deutsche Forschungsgemeinschaft, Bonn, Germany [FOR-753]
Early postmortem changes of porcine muscle proteins including the rate and extent of pH decline, proteolysis and oxidation are key factors influencing the loss of water in meat, and proteolytic degradation may result in shrinking of muscle cells and drip loss. We report here the identification and structural characterisation of post-mortem degradation and oxidation of myofibrillar proteins using high resolution mass spectrometric proteomics. Soluble muscle proteins from M. Longissimus dorsi obtained 48 h postmortem at different drip loss were separated by two-dimensional gel electrophoresis (2D-PAGE), and degradation products were identified by Fourier-transform ion cyclotron resonance mass spectrometry. Oxidation products were detected by 2D-oxyblot analysis of 2,4-dinitrophenylhydrazine (DNPH)-treated proteins using an anti-DNP antibody, and selected spots were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Postmortem denaturation at low drip loss was found for four contractile proteins, myosin-light chain-1; myosin regulatory light chain; alpha-beta-tropomyosin and alpha-actin. The combination of 2D-PAGE and FTICR-MS was found to be a powerful approach for identification of muscle protein degradation products, providing identification of several truncation forms of creatine kinase and troponin T. The comparison of 2D-oxyblot and silver-stained 2D-gels at low and high drip loss revealed approximately 70 oxidatively modified proteins from muscle cell lysate. Oxidative modifications, representing possible biomarker candidates, were identified at Lys-170 of creatine kinase (4-hydroxynonenal), Lys-326 of actin (amino-adipic semialdehyde), and at W-169 (kynurenine) of triosephosphate isomerase. (C) 2010 Published by Elsevier B.V.
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