Proteome analysis as an index of conformational change of protein

在本研究中，我们首先利用模型蛋白来检测蛋白质结合小分子上的构象变化。其次，对合成的巯基标记剂的荧光强度变化进行评价，分析蛋白质的构象变化。该方法还可以利用MALDI-PSD TOF MS分析巯基荧光标记剂标记的多肽中的氨基酸序列，并对蛋白酶消化的荧光标记蛋白进行高效液相色谱（HPLC）分离。合成了两种相似的巯基分子荧光剂，将这两种荧光剂与MALDI-TOF MS结合，通过比较两种荧光剂分别标记后酶切肽的相对信号强度比，可以评价蛋白质的构象变化。另一方面，开发了蛋白质裂解过程中多肽c端标记的新方法。该方法可用于各种带电荷的烷基胺的标记和结构分析，也可用于与MALDI-TOF ms的比较测定。此外，还考察了多胺对丝氨酸蛋白酶消化蛋白质的影响。血红蛋白作为模型蛋白，在多胺存在下用胰蛋白酶消化。产物肽分离，HPLC采集，MALDI-TOF质谱后源衰减分析。结果表明，部分多肽确实在c端被多胺修饰。

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.