IMMUNO-AFFINITY MASS SPECTROMETRY APPLIED TO STUDY OF AMYLOIDOSIS

我们已经启动了一项联合使用免疫亲和技术的研究 用基质辅助激光解吸质谱检测 鉴定P-淀粉样蛋白相关多肽。在本研究中，单克隆 以抗β-淀粉样肽抗体为“鱼钩” 特异、灵敏地捕获目标多肽--淀粉样蛋白 样品中的相关多肽。淀粉样蛋白相关多肽是第一个 抗淀粉样蛋白单抗的免疫沉淀。这个 用蛋白A/琼脂糖沉淀抗体-抗原复合体。 在洗去非特异性结合多肽后，该复合体被 采用基质辅助激光解吸质谱进行分析。这个 这种方法的灵敏度和量化方面都经过了仔细的研究 已评估。我们实验室建立了细胞培养设备，以 促进细胞生物学研究和中药炮制的研究 β-淀粉样蛋白体外培养。我们已经用我们的方法调查了 P-淀粉样蛋白在一系列培养细胞系中的加工 (例如，我们已经研究了S 182基因突变对人类健康的影响 14号染色体对淀粉样蛋白P的产生和降解的影响) 并在不久的将来计划将调查扩展到分析 阿尔茨海默病患者脑脊液中淀粉样多肽的变化。一个 描述这些结果的论文已经发表在《生物》杂志上。 化学。272、43234326、1997年。目前我们正在：分析 洗涤剂在免疫沉淀/质谱分析中的应用 蛋白质。不同洗涤剂对洗涤效果的比较 提取胞内AP多肽。优化洗涤条件 用于去除非特异性结合蛋白和洗涤剂 免疫沉淀。

We have initiated a study using immuno-affmity technique combined with matrix-assisted laser desorption mass spectrometry to characterize P-Amyloid related peptides. In this study, monoclonal anti-p-amyloid peptide antibody was used as a "fishhook" to specifically and sensitively capture the target peptide-amyloid related peptides in samples. Amyloid related peptides was first immunoprecipitated by monoclonal Anti-amyloid antibodies. The antibody-antigen complex was precipitated by protein A/Gagarose. After washing away non-specific bonded peptides, the complex was analyzed by matrix-assisted laser desorption mass spectrometry. The sensitivity and quantitative aspects of this method has been carefully evaluated. Cell culture equipment was set up in our laboratory to facilitate cell biological studies and studies of the processing of P-Amyloid in vitro. We have used our method to investigate the processing of P-Amyloid protein in a series of cultured cell lines (e.g.,we have investigated the effects of the S 182 gene mutation on chromosome- 14 on the production and degradation of amyloid P protein) and in the near future plan to extend the investigation to an analysis of amyloid peptides in CSF of patients with Alzheimer's disease. A paper describing these results has been published in the J. Biol. Chem. 272, 43234326, 1997. Currently we are: Analyze the effect of detergents on immunoprecipitation/mass spectrometry analysis of proteins. Compare the effeciency of different detergents on extracting intracellular AP peptides. Optimize the washing condition for removing non-specific boned protein and detergents used in immunoprecipitation.