質量分析による変異蛋白質の迅速検出と臨床検査への応用

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The soft ionization mass spectrometry such as MALDI-TOF and ESI are powerful tools for the detection of proteins, peptides and other biological molecules in body fluids. Since biologically active compounds occur at very low concentrations in mixtures containing salts, there has arisen the need for some special sample handling techniques. In this study, we described some simple preparations for the detection of abnormal proteins in blood and urine prior to the analysis by MALDI-TOF and ESI mass spectrometry.First, by only dilution with distilled water to 10-150 times, albumin and the incomplete immunoglobulin heavy chain in sera and albumin, Bence Jones protein, β2-microglobulin, hemoglobin and lysozyme in urine from patients with nephritis, monoclonal gammopathy and monocytic leukemia were clearly detected. Second, adsorption of albumin with Blue gel or IgG with Protein G Sepharose 4FF could be improved the sensitivities and signal-to-noise ratios for the detections of these proteins. Finally, by immunoprecipitations with specific antisera, proteins at lower concentrations, such as carbohydrate deficient transferrin, could be easily detected using MALDI-TOFMS as well as on-line HPLC/ESIMS analysis.As to the analysis of variant hemoglobinopathy, the peptides prepared by digestion of intact globin were analyzed and nearly 90 significant peaks were detected by a single spectrum from ESI analysis. Ion peaks which were assigned with some degree of certainly covered whole sequence from a- and b-subunits. Especially, at the analysis of a variant hemoglobin “core”, the free cystein residue would be converted into cysteic acid by performic acid oxidation before enzymatic cleavage, which would increase the recoveries of the peptides in the “core” regions. The tandem mass spectrometry with oxidized globin mixture digested with trypsin and lysyl endopeptidase will offer a standard method to detect substitution in the “core” region of hemoglobin.
日本質量分析学会の論文
1996-06-01

質量分析による変異蛋白質の迅速検出と臨床検査への応用

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