申请人:University of Washington
公开号:US20020076739A1
公开(公告)日:2002-06-20
Analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The methods employ affinity labeled protein reactive reagents having three portions: an affinity label (A) covalently linked to a protein reactive group (PRG) through a linker group (L). The linker may be differentially isotopically labeled, e.g., by substitution of one or more atoms in the linker with a stable isotope thereof. These reagents allow for the selective isolation of peptide fragments or the products of reaction with a given protein (e.g., products of enzymatic reaction) from complex mixtures. The isolated peptide fragments or reaction products are characteristic of the presence of a protein or the presence of a protein function in those mixtures. Isolated peptides or reaction products are characterized by mass spectrometric (MS) techniques. The reagents also provide for differential isotopic labeling of the isolated peptides or reaction products which facilitates quantitative determination by mass spectrometry of the relative amounts of proteins in different samples. The methods of this invention can be used for qualitative and quantitative analysis of global protein expression profiles in cells and tissues, to screen for and identify proteins whose expression level in cells, tissue or biological fluids is affected by a stimulus or by a change in condition or state of the cell, tissue or organism from which the sample originated.
分析试剂和基于质谱的方法,使用这些试剂对蛋白质或蛋白质功能在蛋白质混合物中进行快速和定量分析。该方法采用亲和标记蛋白质反应试剂,具有三个部分:亲和标记(A)通过连接基团(L)共价连接到蛋白质反应基团(PRG)。连接基团可以通过用稳定同位素替换连接基团中的一个或多个原子而不同地同位素标记。这些试剂允许从复杂混合物中选择性地分离肽片段或与给定蛋白质的反应产物(例如酶反应产物)。分离的肽片段或反应产物特征显示存在于这些混合物中的蛋白质或蛋白质功能的存在。分离的肽片段或反应产物通过质谱技术进行表征。这些试剂还可提供对分离的肽片段或反应产物进行不同同位素标记,从而便于通过质谱法定量测定不同样品中蛋白质的相对量。本发明的方法可用于细胞和组织中全局蛋白质表达谱的定性和定量分析,以筛选和识别受刺激或细胞、组织或生物体状态或条件变化影响其表达水平的蛋白质在细胞、组织或生物体液中的存在。