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

在后基因组时代，了解基因组在细胞、组织和生理体液中表达蛋白质的功能是非常重要的。蛋白质的双向电泳图谱（2D SDS-PAGE）为a。用于分离和表征基因组表达的蛋白质的有用工具。它也可能被用作诊断工具，如特定疾病的“标记”蛋白。组织中蛋白质的表征有时难以完成，特别是对于低拷贝数的蛋白质。本研究利用ID/ 2D SDS-PAGE技术，对细胞或组织细胞器中的蛋白表达谱进行了分析，并将其作为临床分子扫描仪。首先，我们发展了一种新的程序，使用冻融到Nycodenz的密度梯度溶液的一步分离的细胞器从大鼠肝脏。Nycodenz的密度梯度是从20%的溶液在离心管中通过冷冻-解冻过夜-20 ℃和室温下几小时制备的。 ...更多信息 没有初始离心程序。将去除细胞核和胞质组分的大鼠肝匀浆在Nycodenz梯度溶液上分层，离心并分离亚细胞组分。分级分离后，使用ID SDS-PAGE检查蛋白质谱。使用Western印迹法确认每个细胞器的标记蛋白。在2D SDS-PAGE上对大鼠肝脏主要组分中的表达蛋白进行了分离。蛋白质中半胱氨酸残基的还原和烷基化是蛋白质组分析凝胶消化前的重要步骤。我们开发了在SDS凝胶电泳过程中与丙烯酰胺原位烷基化，得到硫醚衍生物，cys-S-β-丙酰胺（PAM-cys）。半胱氨酰肽和总胰蛋白酶肽的覆盖度增加。PAM-cys在数据库中易于识别。采用细胞器分级分离、ID/2D SDS-PAGE等方法对成年和老年小鼠肝细胞器蛋白质表达谱进行了比较。少

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