Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.
Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.
复制标题
使用连续肽亲和纯化结合串联质谱法鉴定大肠杆菌中的蛋白质复合物。
DOI：
--
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
A. Emili
中科院分区：
文献类型：
--
作者：
M. Babu;O. Kagan;Hongbo Guo;J. Greenblatt;A. Emili
由于大多数细胞过程都是由大分子组装介导的，因此系统地识别蛋白质-蛋白质相互作用(PPI)和识别多蛋白质复合体的亚基组成可以提供对基因功能的洞察和对生物系统的理解(1，2)。基于染色体标记蛋白质的亲和纯化结合质谱学(APMS)，可以在近生理条件下以高可信的大规模分离和表征内源蛋白质复合体来描绘物理相互作用。这种方法已经成功地应用于进化上不同的生物，包括酵母、苍蝇、蠕虫、哺乳动物细胞和细菌(1-6)。特别是，我们已经产生了一个羧基末端序列多肽亲和力(SPA)双重标签系统，用于从培养的革兰氏阴性大肠杆菌中亲和纯化天然蛋白质复合体，使用基因易处理的宿主实验室菌株，这些菌株非常适合于原核生物的基础生物学和保守过程的全基因组研究(1，2，7)。我们的SPA标签系统类似于最初为酵母(8，9)开发的串联亲和纯化方法，由一个钙调蛋白结合肽(CBP)和高度特异的烟草蚀刻病毒(TEV)蛋白酶的切割位点和三个FLAG表位(3X FLAG)组成，允许连续两轮亲和浓缩。在盒扩增之后，编码SPA标签和可选标记的序列特异性线性PCR产物被整合并在DY330背景中以羧基末端融合的形式在框架中表达，该融合被诱导以瞬时表达高效的异源噬菌体lambda重组系统(10)。随后使用钙调蛋白和抗FLAG亲和珠的两步纯化使大规模培养中即使是低丰度的蛋白质复合体也能够高度选择性和高效地回收。然后使用串联质谱仪以高灵敏度(低纳克检测下限)鉴定稳定共纯化的蛋白质。在这里，我们描述了我们通常用于系统的蛋白质标记、纯化和基于质谱分析的大肠杆菌可溶性蛋白质复合体的详细步骤，这些蛋白质复合体可以扩大规模，并有可能针对其他细菌物种量身定做，包括某些易于重组的机会性病原体。由此产生的物理相互作用通常可以揭示有趣的意想不到的组件和连接，暗示着新的机械联系。将PPI数据与交替的分子关联数据(如遗传(基因-基因)相互作用和基因组上下文(GC)预测)相结合，可以帮助阐明生物途径中多蛋白质复合体的全球分子组织。为大肠杆菌产生的网络可以用来洞察目前缺乏功能注释的其他微生物中的同源基因产物的功能结构。
Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria(1-6). In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes(1, 2, 7). Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast(8, 9), and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system(10). Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits). Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.