Fumio Nomura1)2)3), Kazuyuki Sogawa2), Yoshio Kodera2)4), Hiroshi Umemura1), Motoi Nishimura1),Mamoru Sato2), Setsu Sawai1), Kazuyuki Matsushita1), Osamu Yokosuka3), Kenta Noda5), Iwao Kiyokawa5), Toshihide Miura5), Ryo Kojima5), Katsuhiro Katayama5)
1)Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 2)Division of Clinical Genetics and Proteomics, Chiba University Hospital, 3)Division of Gastroenterology, Chiba University Hospital,4)Department of Physics, Kitasato University School of Science, 5)Medical Development Center, Nittobo Medical Co., LTD
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Recent progress in proteomic technologies allows us to detect and identify a number of novel biomarker candidates. However, there are challenges in translating clinical proteomics from bench to bedside. We previously detected and identified novel biomarker candidates in heavy drinkers by SELDI-TOF MS(1) and more recently by the MALDI-TOF /TOF MS(2). Among several marker candidates, the 5.9 kDa peptide identified as a fragment of fibrinogen alpha chain C-terminal (FIC 5.9) appears most promising. To bring FIC 5.9 measurement to a level with potential for use in clinical diagnostics, we tried to establish a simple and reproducible system by combining the ClinProtTM System and a stable isotope-labeled peptide standard (SI-MS).
A total of 100 serum samples obtained from patients with alcoholic liver diseases, chronic hepatitis C, liver cirrhosis and apparently healthy volunteers were analyzed. Serum FIC 5.9 levels were determined by the ClinProtTM system using weak cationic exchange beads and the AutoFlex® TOF/TOF mass spectrometer(Bruker Daltonics). Synthetic FIC 5.9 was isotope labeled with 13C and 15N and was used as an internal standard for quantification of FIC 5.9.
Overall imprecision was 4.6%-15.8% by the conventional way without the isotope-labeled standard, whereas those for the SI-MS was 2.1%-6.2%. Serum FIC 5.9 levels significantly changed depending on the time intervals between venipuncture and serum separation; the level increased in a time-dependent manner until 30 min and then remained stable up to 4 hrs. It was essential to keep the samples at room temperature for at least 30 min before obtaining sera. Influences of freeze-thaw cycles were minimal. Serum levels of FIC 5.9 levels in healthy subjects were 2.0}0.2 AU/ml(Mean}SD),whereas those for patients with alcoholic liver diseases were 1.05}0.63 (p<0.01). Interestingly, FIC 5.9 levels were even lower in patients with chronic hepatitis C (0.74}0.17 ; P<0.001),indicating that FIC 5.9 could be a sensitive biomarker for chronic liver injury with broad-spectrum pathology.
Thus, this simple and reproducible system combining ClinProtTM System and a stable isotope-labeled peptide standard enabled us to standardize preanalytical factors and to further elucidate clinical implications of FIC 5.9 measurement. We are planning to compare these data with those to be obtained by enzyme immunoassays for FIC 5.9, which we could develop very recently.
(1) Proteomics 2004; 4: 1187-1194
(2) Proteomics Clin Appl 2009; 3: 821-828 |
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