MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting
MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting
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DOI：
10.1074/mcp.o117.067082
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发表时间：
2018-02-01
影响因子：
7
通讯作者：
Shevchenko, Andrej
中科院分区：
文献类型：
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作者：
Kumar, Mukesh;Joseph, Shai R.;Shevchenko, Andrej
蛋白质的绝对定量阐明了多蛋白质组装体和网络的分子组成、调控和动力学。在这里，我们报告了一种称为MS Western的方法，该方法可以准确地确定全细胞或组织裂解物中亚飞摩尔水平的数十种用户选择的蛋白质的摩尔丰度，而无需代谢或化学标记，也无需使用特异性抗体。MS Western依赖于GeLC-MS/MS，并通过凝胶内共消化与同位素标记的QconCAT蛋白质嵌合体（由串联的蛋白质型肽组成）定量蛋白质。它不需要对嵌合体进行纯化，并且将所有蛋白质型肽的摩尔丰度与单个参考蛋白质相关联。在比较实验中，MS Western在蛋白质检测特异性、线性动态范围和蛋白质定量灵敏度方面优于免疫荧光Western印迹。为了在体内实验中验证MS Western，我们在早期胚胎发生的十个阶段期间定量了斑马鱼核心组蛋白H2 A、H2 B、H3和H4的摩尔含量。准确定量（CV
Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV