2-D LC-MS/MS Analysis of secreted proteins from HepG2 cells : Combination with various sample preparation methods before in-solution trypsin digestion
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概要
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Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS) technique has high capability of resolving peptides, and is used for analysis of tryptic peptides derived from highly complex protein mixtures such as plasma. However, the detection of low-abundant proteins and low-molecular-weight proteins is often very difficult in this system, because major peptides from high-abundant proteins such as albumin mostly disturb the separation of minor peptides from low-abundant proteins. To resolve these problems, different methods of sample preparation for in-solution trypsin digestion were tested, and the optimized method was applied to the 2-D LC-MS/MS analysis of proteins secreted from human hepatoma cell line, HepG2. We could identify 247 and 142 proteins from the chemically and thermally denatured samples, respectively. Among them, 71 proteins were identified in common to both methods, while most of the proteins were identified using either of the two procedures. In addition to these denaturation methods, the molecular-mass cutoff via ultrafiltration improved the efficiency in identifying low-molecular-weight proteins. Finally, we could identify 478 secreted proteins in total using the combination of these processes. These data indicate that in sample preparation the combination of various denaturation methods, as well as molecular-mass cutoff, are very critical for the identification of a wider range of low-abundant proteins via 2-D LC-MS/MS analysis.
- 2005-03-01
著者
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YASUDA Kazuki
Department of Metabolic Disorder, Research Institute, International Medical Center of Japan
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Yasuda Kazuki
Department Of Metabolic Disorder Research Institute International Medical Center Of Japan
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Yasuda Kazuki
Advanced Medical Research Laboratories Tanabe Seiyaku Co. Ltd.
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Kamisaka Yasushi
Advanced Medical Research Laboratories Tanabe Seiyaku Co. Ltd.
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YAMASHITA RYO
Department of Endocrinology and Metabolism, Yokohama City University Graduate School of Medicine
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Fujiwara Yuko
Department Of Metabolic Disorder Research Institute International Medical Center Of Japan
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Yasuda Kazuki
Department Of Internal Medicine And Molecular Science Graduate School Of Medicine Osaka University
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Kaburagi Yasushi
Department Of Internal Medicine And Molecular Science Graduate School Of Medicine Osaka University
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Kaburagi Yasushi
Advanced Medical Research Laboratories Tanabe Seiyaku Co. Ltd.
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YUAN Xunmei
Department of Metabolic Disorder, Research Institute, International Medical Center of Japan
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Fujiwara Yuko
Department Of Health And Sports Science Faculty Of Health Science And Technology Kawasaki University
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Yamashita Ryo
Department Of Metabolic Disorder Research Institute International Medical Center Of Japan
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Yuan Xunmei
Department Of Metabolic Disorder Research Institute International Medical Center Of Japan
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Yamashita Ryo
Department Of Endocrinology And Metabolism Yokohama City University Graduate School Of Medicine
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Kaburagi Yasushi
Department Of Diabetic Complications Diabetes Research Center Research Institute National Center For Global Health And Medicine
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Yasuda Kazuki
Department of Endocrinology and Metabolism, International Medical Center of Japan
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