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2D gel separation and sequence analysis of protein in micro amount : novel C-terminal sequencing methods.

微量蛋白质的二维凝胶分离和序列分析：新型C端测序方法。

基本信息

批准号：
04102008
负责人：
TSUGITA Akira
金额：
$ 45.44万
依托单位：
SCIENCE UNIVERSITY OF TOKYO,RESEARCH INSTITUTE OF BIOSCIENCES
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

1) Novel C-terminal sequencing : Vapor from 90% perfluoroic acid aqueous solution on peptides at 90ﾟC for 4-24 hours results mixture of C-terminal truncate molecules. FAB-or ESI-mass spectrometry can alnalyze these C-terminal truncate degradated molecular ions. The mass differences of truncate ions directly give the C-terminal sequence. The reaction accompanies internal cleavage at C-terminal side of Asparatic acid and N-terminal side of serine. Vapor from 15-30% perfluoacyl anhydride on peptides-20ﾟC for 1 hour also results the mixture of C-terminal degraded molecules, analyzed by FAB-mass spectrometry as truncate molecular ions. Accompanying major degraded ions, -1, -18, -46 ions were observed and these ions were experimentally identified. The reaction mechanism was partly elucidated. Conditions of the reproducible experiments were extensively studied and the entire process to be in the glove box with N2 flow to avoid moisture at low temperature. Further more study is needed to big m … More olecule and automation of the method.2) N-terminal sequencing : Hetrogeneous reaction system is found to be favor for micro-sequencing chemical reaction. The intermediate ATZ compound (solid) in Edman degradation was amidated by gas and this reaction yields were found still 70% in femto mole range. According to this system, alternate amidation with two fluorescence-amines has been studied. A half of the native proteins waas found blocked at their N-termini. Anhydrous hydrazine vapor was found to be used for deblocking formyl-and pyroglutamyl residues but not deblock acetyl and acyl groups at N-termini, which is under progress.3) 2D-gel electrophoresis : A new concept of standardization was proposed with use of commercially available reference proteins. Three biological systems were used : rice, Arabidopsis and Fusarium (fungi). Numbers of separated protein spots were about 5,000,5,000 and 1,500 using the dimension, 20x20cm. The numbers of 5,000 may be maximum and may be one third or a half of these plant entire proteins. In Fusarium all proteins can be estimated to be 1,500. Partial N-terminal sequencings were carried out about 100-150 proteins for the respective system, and a half of them were sequenced and the other half were blocked at their N-termini. Altogether 70 proteins were carried out by our new deblocking method and more than one tenth were deblocked and sequenced. The approach with 2D may be complementary used for genome research. A novel homology research method was proposed. Less

1)新型C端测序：90%全氟酸水溶液在90°C下蒸发4-24小时，得到C端截断分子混合物。fab -或esi -质谱法可以分析这些c端截断降解的分子离子。截断离子的质量差直接给出了c端序列。该反应伴随着天冬氨酸c端和丝氨酸n端的内部裂解。15-30%的全氟酸酐在肽-20 - C上蒸发1小时也会产生C端降解分子的混合物，通过fab -质谱分析为截断分子离子。同时观察到主要的降解离子-1、-18、-46，并对这些离子进行了实验鉴定。部分阐明了反应机理。对重复性实验的条件进行了广泛的研究，整个过程在手套箱中进行N2流动，以避免低温受潮。该方法的分子化程度和自动化程度有待进一步研究。2) n端测序：发现非均相反应体系有利于微测序化学反应。Edman降解的中间ATZ化合物（固体）被气体修饰，该反应收率在femto - mol范围内仍为70%。根据该体系，研究了两种荧光胺的交替酰胺化反应。一半的天然蛋白被发现在它们的n端被阻断。无水肼蒸汽可用于甲酰和焦谷氨酰基残基的解封，但不能用于n端乙酰基和酰基的解封，这一研究还在进行中。3) 2d凝胶电泳：利用市售参考蛋白，提出了标准化的新概念。采用三种生物系统：水稻、拟南芥和镰刀菌。使用尺寸为20x20cm，分离的蛋白点数量分别为5000、5000和1500个。5000的数量可能是最大的，可能是这些植物全部蛋白质的三分之一或一半。在镰刀菌中，所有的蛋白质估计有1500个。对各系统约100-150个蛋白进行了部分n端测序，其中一半被测序，另一半在其n端被阻断。我们的新解封方法共对70个蛋白质进行了解封和测序，超过十分之一的蛋白质被解封和测序。2D方法可作为基因组研究的补充。提出了一种新的同源性研究方法。少