Streamlined analysis schema for high-throughput identification of endogenous protein complexes
Streamlined analysis schema for high-throughput identification of endogenous protein complexes
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DOI：
10.1073/pnas.0912599106
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发表时间：
2010-02-09
影响因子：
11.1
通讯作者：
Qin, Jun
中科院分区：
文献类型：
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作者：
Malovannaya, Anna;Li, Yehua;Qin, Jun
免疫沉淀后质谱（IP/MS）最近已成为蛋白质复合物组分和细胞蛋白质网络分析的首选方法。靶向高等真核生物的内源性蛋白质复合物，特别是在大规模的努力，一直具有挑战性，由于细胞的异质性，高蛋白质组的复杂性，以及相比于较低的生物体，缺乏有效的位点内表位标记技术。非特异性鉴定的变异性和一抗的交叉反应性使其进一步复杂化。尽管如此，内源性人类蛋白质网络的研究是非常需要的，尽管它的挑战。在这里，我们描述了一个精简的IP/MS协议的纯化和鉴定扩展的内源性蛋白质复合物。我们调查来源的非特异性蛋白结合和开发半定量特异性过滤器，是基于肽光谱计数测量。我们还概述了从IP/MS数据推导准确的复合物组成的逻辑约束，并通过介绍我们对不同转录辅调节因子复合物的分析来证明这种方法的有效性。我们一致纯化的新组件的集成商复杂，分析的中介复杂的组合物，仅从我们的数据来证明光谱计数的广泛可用性，和deconvolute异构HDAC 1/2网络到核心复杂的模块和几个新的subcomplex相互作用。
Immunoprecipitation followed by mass spectrometry (IP/MS) has recently emerged as a preferred method in the analysis of protein complex components and cellular protein networks. Targeting endogenous protein complexes of higher eukaryotes, particularly in large-scale efforts, has been challenging due to cellular heterogeneity, high proteome complexity, and, compared to lower organisms, lack of efficient in-locus epitope-tagging techniques. It is further complicated by variability in nonspecific identifications and cross-reactivity of primary antibodies. Still, the study of endogenous human protein networks is highly desired despite its challenges. Here we describe a streamlined IP/MS protocol for the purification and identification of extended endogenous protein complexes. We investigate the sources of nonspecific protein binding and develop semiquantitative specificity filters that are based on peptide spectral count measurements. We also outline logical constraints for the derivation of accurate complex composition from IP/MS data and demonstrate the effectiveness of this approach by presenting our analyses of different transcriptional coregulator complexes. We show consistent purification of novel components for the Integrator complex, analyze the composition of the Mediator complex solely from our data to demonstrate the wide usability of spectral counts, and deconvolute heterogeneous HDAC1/2 networks into core complex modules and several novel subcomplex interactions.