公开(公告)号：US07045296B2

公开(公告)日：2006-05-16

申请号：US09851058

申请日：2001-05-08

申请人： Kenneth C. Parker ,  Timothy K. Nadler ,  George J. Vella ,  Yulin Huang ,  Rudolf H. Aebersold ,  Marcus B. Smolka

发明人： Kenneth C. Parker ,  Timothy K. Nadler ,  George J. Vella ,  Yulin Huang ,  Rudolf H. Aebersold ,  Marcus B. Smolka

IPC分类号： G01N33/53 ,  G01N24/00

CPC分类号： G01N33/6803 ,  Y10S435/964 ,  Y10T436/24 ,  Y10T436/25125

摘要： Methods using gel electrophoresis and mass spectrometry for the rapid, quantitative analysis of proteins or protein function in mixtures of proteins derived from two or more samples in one unit operation are disclosed. In one embodiment the method includes (a) preparing an extract of proteins from each of at least two different samples; (b) providing a set of substantially chemically identical and differentially isotopically labeled protein reagents; (c) reacting the extract of proteins from different samples of step (a) with a different isotopically labeled reagent from the set of step (b) to provide two or more sets of isotopically differentially labeled proteins; (d) mixing each of the two or more sets of isotopically labeled proteins to form a single mixture of isotopically differentially labeled proteins; (e) electrophoresing the mixture of step (d) by an electrophoresing method capable of separating proteins within the mixture; (f) digesting at least a portion of one or more separated proteins of step (e) and (g) detecting the difference in the expression levels of the proteins in the two samples by mass spectrometry based on one or more peptides in the sample of labeled peptides. The analytical method can be used for qualitative and particularly for quantitative analysis of global protein expression profiles in cells and tissues, i.e. the quantitative analysis of proteomes.

摘要翻译： 公开了使用凝胶电泳和质谱法在一个单元操作中从两个或更多个样品衍生的蛋白质的蛋白质或蛋白质功能的快速，定量分析的方法。 在一个实施方案中，该方法包括（a）从至少两种不同样品中的每一种制备蛋白质提取物; （b）提供一组基本上化学相同和差异同位素标记的蛋白质试剂; （c）使来自步骤（a）的不同样品的蛋白质提取物与来自步骤（b）的组的不同的同位素标记的试剂反应，以提供两组或更多组同位素差异标记的蛋白质; （d）混合两组或更多组同位素标记的蛋白质以形成同位素差异标记的蛋白质的单一混合物; （e）通过能够分离混合物内的蛋白质的电泳方法电泳步骤（d）的混合物; （f）消化步骤（e）中的一种或多种分离的蛋白质的至少一部分，和（g）基于样品中的一种或多种肽通过质谱法检测两种样品中的蛋白质的表达水平的差异 标记肽。 分析方法可用于定性，特别是定量分析细胞和组织中的全球蛋白表达谱，即蛋白质组的定量分析。