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Sequential analysis of expressed proteins in organelles from rat liver using 1D/2D SDS-PAGE as a clinical molecullar scanner

使用 1D/2D SDS-PAGE 作为临床分子扫描仪对大鼠肝脏细胞器中表达的蛋白质进行序列分析

基本信息

批准号：
12670122
负责人：
MURAYAMA Kimie
金额：
$ 1.92万
依托单位：
JUNTENDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

In the post-genome era, it is important to understand the function of genomes, which express the proteins in cells, tissues and physiological fluids. The two-dimensional electrophoresis profile (2D SDS-PAGE) of proteins is a. useful tool for separation and characterization ofproteins expressed by genomes. It also might be used for a diagnostic tool such as " marker" proteins for specific disease. Characterization of proteins in tissues is some times difficult to accomplish particularly for proteins in low copy numbers. In the purpose of our study, the expressed protein profiles in organelles of cell or tissues using the ID/ 2D SDS-PAGE were used as a clinical molecular scanner. First we developed a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver. The density gradient of Nycodenz was prepared from 20% solution in a centrifuge tube by freezing-thawing overnight -20C and at room temperature for a few ho … More urs without the initial centrifugation procedure. Rat liver homogenate, which was removed nuclei and cytosolic fractions, was layered on the Nycodenz gradient solution, centrifuged and separated the subcellular fractions. After fractionation, the protein profile were examined using ID SDS-PAGE. The marker protein of each organelle was confirmed using Western blotting. The expressed proteins in the main fractions of the rat liver were separated on 2D SDS-PAGEs. Reduction and alkylation of cysteinyl residues in proteins is an important process before in gel digestion for proteome analysis. We developed in situ alkylation with acrylamide during SDS gel electrophoresis to yield a thioether derivative, cys-S-beta-propionamide(PAM-cys). The coverage of cysteinyl peptides and total tryptic peptides was increased. And PAM-cys was easy to identify proteins using the databases. The comparison of expressed protein profiles in liver organelles between adult and aging mouse was started by sequential analysis using organelle fractionation, ID/2D SDS-PAGE. Less

在后基因组时代，基因组表达细胞、组织和生理体液中的蛋白质，了解基因组的功能非常重要。蛋白质的二维电泳图谱（2D SDS-PAGE）是一种有用的工具，用于分离和表征基因组表达的蛋白质。它也可能用于诊断工具，如特定疾病的“标记”蛋白质。组织中蛋白质的表征有时很难完成，特别是对于低拷贝数的蛋白质。在我们的研究中，使用ID/ 2D SDS-PAGE在细胞或组织细胞器中表达的蛋白谱作为临床分子扫描仪。首先，我们开发了一种利用Nycodenz密度梯度溶液冷冻解冻一步分离大鼠肝脏细胞器的新方法。在不进行初始离心的情况下，将20%的溶液放入离心管中，在-20℃冻融过夜并在室温下放置几小时，得到Nycodenz的密度梯度。大鼠肝脏匀浆取去核和胞浆部分，分层于Nycodenz梯度液上，离心分离亚细胞部分。分离后，用ID SDS-PAGE检测蛋白谱。Western blotting检测各细胞器的标记蛋白。在2D SDS-PAGEs上分离大鼠肝脏主要部位的表达蛋白。蛋白质中半胱氨酸残基的还原和烷基化是蛋白质组学分析中凝胶消化前的一个重要过程。我们在SDS凝胶电泳过程中与丙烯酰胺原位烷基化，得到了硫醚衍生物cys- s- β -丙酰胺（PAM-cys）。半胱氨酸肽和总色氨酸肽的覆盖率增加。PAM-cys易于通过数据库识别蛋白质。利用细胞器分离技术（ID/2D SDS-PAGE）进行序列分析，比较成年小鼠和老年小鼠肝细胞器中表达的蛋白谱。少