HIGHLY SPECIFIC ANALYTICAL METHOD FOR SMALL MOLECULE-TARGETED PROTEINS

小分子靶向蛋白质的高度特异性分析方法

基本信息

批准号：
17590029
负责人：
MANO Nariyasu
金额：
$ 2.3万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

I developed a new specific extraction method for capturing small molecule-binding proteins from biological fluids by the cleavable affinity gel. This gel has the small molecule ligand immobilized onto the agarose gel via the disulfide linker, and the captured proteins can be extracted to the supernatant as the ligand-protein complex by reductive cleavage reaction without any conformational change of proteins.In the model experiment, this method was applied to extraction of deoxycholate-binding proteins in mouse ascites containing anti-deoxycholate monoclonal antibody. In SDS-PAGE, three protein bands were observed in the extraction fraction, and the mass spectrometric analysis and database search following in-gel tryptic digestion indicated that they were serum albumin and heavy chain and light chain of anti-deoxycholate monoclonal antibody.Next, I tried to extract the chenodeoxycholate-binding proteins in the rat brain tissue. The cytoplasmic fraction of cerebrum, midbrain, cerebellum, brainstem, hippocampus, and pituitarium were separately mixed to the chenodeoxycholate-immobilized cleavable affinity gel, and the captured proteins were analyzed by SDS-PAGE. All tissues contain three protein bands in the extraction fraction. After in-gel tryptic digestion and analysis by MALDI-TOF MS, they were identified as α-, β-tubulin, β-actin, and 14-3-3 protein by the database search. Moreover, two abundant protein bands were observed in the extraction fraction from pituitarium, and identified as serum albumin and growth hormone by MALDI-TOF MS and database search. Serum albumin is one of the carrier proteins of bile acids, and plays an important role in bile acid transport in blood stream. The extraction of serum albumin indicated that this extraction method was useful for specifically capturing small molecule-binding proteins from biological fluids.

我开发了一种新的特异性提取方法，用于通过可裂解的亲和力凝胶从生物液体中捕获小分子结合蛋白。该凝胶具有通过二硫键连接器固定在琼脂糖凝胶上的小分子配体，并且可以将捕获的蛋白质提取到上清液中，因为配体 - 蛋白质复合物通过减少蛋白质的裂解反应，而无需对蛋白质的任何构型变化。单克隆抗体。在SDS-PAGE中，在提取部分中观察到了三个蛋白质条，并且质谱分析和数据库搜索后，凝胶胰蛋白酶内消化后，它们是血清白蛋白，抗脱氧乙酸单克基单克酮抗体的重链和轻型链。大脑，中脑，小脑，脑干，海马和puputiaturium的细胞质分数分别混合到Chenodeoxyoxycy硫酸盐含量的可裂解亲和力凝胶，并通过SDS-PAGE分析捕获的蛋白质。所有组织在提取部分中包含三个蛋白质带。通过MALDI-TOF MS进行凝胶胰蛋白酶的消化和分析后，通过数据库搜索鉴定为α-，β-微管蛋白，β-肌动蛋白和14-3-3蛋白。此外，在从Pitutiaturium的提取分数中观察到了两个丰富的蛋白质带，并通过MALDI-TOF MS和数据库搜索被鉴定为血清白蛋白和生长曲。血清白蛋白是胆汁酸的载体蛋白之一，在血流中的胆汁酸转运中起着重要作用。血清白蛋白的提取表明，这种提取方法可用于特异性地捕获来自生物流体的小分子结合蛋白。