Establishment and application of a high-quality comparative analysis strategy for the discovery and small-scale validation of low-abundance biomarker peptides in serum based on an optimized novel peptide extraction method
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概要
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Low-abundance native peptides are an attractive target for the discovery of disease biomarkers. However, validating candidate peptides is difficult due to challenges associated with precise peptide identification and development of high-throughput assays using specific antibodies. Therefore, a highly reproducible and sensitive strategy based on effective peptide enrichment methods is needed to identify clinically useful biomarkers. We optimized our novel differential solubilization (DS) peptide extraction method to selectively enrich peptides less than 6 kDa, using tricine-SDS-PAGE to evaluate the optimization. The modified DS method was combined with liquid chromatography-mass spectrometry (LC-MS) using conventional high-performance liquid chromatography (HPLC). The reproducibility and sensitivity of the proposed strategy were sufficient to enable discovery of low-abundance (ng/mL range) candidate biomarker peptides. A total of 40 serum samples collected pre- and post-surgery from renal cell carcinoma (RCC) patients were analyzed, resulting in discovery of 2 peptides that are upregulated and one peptide that is downregulated in pre-surgery RCC patients. These peptides were validated using 40 serum samples collected pre- and post-surgery from bladder tumor (BT) patients. Two candidate peptides that were upregulated in pre-surgery RCC patients were not upregulated in the sera of the pre-surgery BT patients. Finally, we propose 2 candidate marker peptides that could be used to detect RCC.
著者
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Satoh Mamoru
Clinical Proteomics Research Center Chiba University Hospital
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KAWASHIMA Yusuke
Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science
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Maeda Tadakazu
Laboratory Of Biomolecular Dynamics Department Of Physics Kitasato University School Of Science
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Minamida Satoru
Department Of Urology Kitasato Institute Hospital
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Nomura Fumio
Department Of Molecular Diagnosis Chiba University Graduate School Of Medicine
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Iwamura Masatsugu
Department Of Urology Kitasato University Hospital
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Kawashima Yusuke
Laboratory Of Biomolecular Dynamics Department Of Physics Kitasato University School Of Science
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Matsui Takashi
Laboratory Of Exercise Biochemistry And Neuroendocrinology University Of Tsukuba Graduate School Of Comprehensive Human Sciences
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Baba Shiro
Department Of Neurosurgery Nagasaki University Graduate School Of Biomedical Sciences
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SATOH Mamoru
Clinical Proteomics Research Center, Chiba University Hospital
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Saito Tatsuya
Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science
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Matsumoto Kazumasa
Center for Disease Proteomics, Kitasato University School of Science
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Araki Keita
Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science
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Kodera Yoshio
Clinical Proteomics Research Center, Chiba University Hospital
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Nomura Fumio
Department of Molecular Diagnosis (F8) Graduate School of Medicine, Chiba University
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Matsui Takashi
Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science
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