Proteome analysis as an index of conformational change of protein

蛋白质组分析作为蛋白质构象变化的指标

基本信息

批准号：
16590033
负责人：
SHIRAHATA Akira
金额：
$ 2.3万
依托单位：
Josai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

In this study, firstly we developed the detection for conformational change of proteins on their binding small molecules by using a model protein. Secondly, the evaluation of the fluorescent intensity change of synthesized labeling agents for sulfhydryl group enabled the analysis for conformational change of proteins. The method also made it possible to analyze amino acid sequences in peptides labeled with the fluorescent labeling agent for sulfhydryl group by MALDI-PSD TOF MS, following fractionation with high performance liquid chromatography (HPLC) for the fluorescence-labeled proteins digested by protease.Two similar molecular fluorescent agents for sulfhydryl group were synthesized, and the combination of the two agent and MALDI-TOF MS enabled the evaluation for conformational changes of proteins, by comparison of relative signal intensity ratio of between the peptides digested following label of the two agents respectively.On the other hand, the novel labeling method for C-terminal labeling in the peptides during cleavage of proteins was developed. This method is found to be applicable for labeling by various alkylamines with some charges, and for structural analysis, and also for comparison determination with MALDI-TOF MS.Furthermore, the effect of polyamines on the digestion of proteins by serine proteases was examined. Hemoglobin was used as a model protein and was digested with trypsin in the presence of polyamine. The product peptides were separated, collected by HPLC, and analyzed by MALDI-TOF MS using post-source decay. The results showed that some peptides were indeed modified with polyamine at the C-terminus.

在这项研究中，首先，我们通过使用模型蛋白来开发出蛋白质在其结合小分子上构象变化的检测。其次，评估硫赖尔组合成标记剂的荧光强度变化使得蛋白质构象变化的分析。该方法还使得可以通过MALDI-PSD TOF MS分析用MALDI-PSD TOF MS的荧光标记剂标记的肽中的氨基酸序列，在用高性能液相色谱（HPLC）分馏后，用于荧光标记的蛋白质的荧光标记蛋白质和蛋白质的两种类似分子荧光经纪人的组合。通过比较蛋白质的肽之间的相对信号强度比分别比较了两种剂的标记，而在两种剂的标记下，蛋白质之间的相对信号强度比分别比较了蛋白质的构象变化。另一方面，开发了C末端标记的新型标记方法。发现该方法适用于通过某些电荷的各种烷基胺和结构分析的标记，以及用于与MALDI-TOF MS.FURTHERMORE进行比较确定，多胺对丝氨酸蛋白酶对蛋白质消化的影响进行了检查。血红蛋白用作模型蛋白，并在多胺存在下用胰蛋白酶消化。分离产物肽，通过HPLC收集，并使用后源衰减通过MALDI-TOF MS分析。结果表明，某些肽确实在C末端用多胺修饰。