Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.
Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.
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DOI：
10.1074/mcp.m113.032367
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发表时间：
2013-12
期刊：
影响因子：
--
通讯作者：
Lamond AI
中科院分区：
文献类型：
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作者：
Kirkwood KJ;Ahmad Y;Larance M;Lamond AI
蛋白质形成多种复合物，介导细胞功能和调节。这种蛋白质复合物的一个很大程度上未探索的特征是特定蛋白质同种型和/或后修饰形式的选择性参与。在这项研究中，我们结合天然尺寸排阻色谱（SEC）与高通量蛋白质组学分析，以表征可溶性蛋白复合物分离自人骨肉瘤（U2 OS）细胞。使用这种方法，我们已经确定了超过71，500肽和1，600磷酸化位点，对应于超过8，000种蛋白质，分布在40个SEC级分中。这代表预测的U2 OS细胞蛋白质组的>50%，以每个蛋白质27%的平均肽序列覆盖率鉴定。进行三次生物学重复，允许对数据进行统计学评价，并证明SEC分馏程序具有高度的重现性。检测到特定的蛋白质与多个独立的复合物相互作用，如MRFAP 1-MORF 4L 1-MRGBP相互作用网络的不同复合物的分离所代表的。这些数据还揭示了选择性地与蛋白质复合物的不同子集相关的蛋白质亚型和翻译后修饰。令人惊讶的是，有明确的富集与差异大小类的蛋白质复合物相关的特定基因本体论术语。这项研究表明，联合SEC/MS分析可用于蛋白质复合物的系统范围内的注释，并预测潜在的异构体特异性相互作用。所有这些SEC关于蛋白质复合物天然分离的数据都已整合到蛋白质组动力学百科全书中，这是一个在线的，多维度的数据共享资源。
Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.