In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.
In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.
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DOI：
10.1074/mcp.m110.002386
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发表时间：
2011-06
期刊：
影响因子：
--
通讯作者：
Lilley KS
中科院分区：
文献类型：
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作者：
Rees JS;Lowe N;Armean IM;Roote J;Johnson G;Drummond E;Spriggs H;Ryder E;Russell S;St Johnston D;Lilley KS
亲和纯化与质谱联用为鉴定蛋白质及其结合伴侣提供了可靠的方法。在这项研究中，我们使用果蝇蛋白三重标记的旗帜，链球菌II，黄色荧光蛋白在体内的亲和下拉实验和分离这些蛋白质在其原生复合物从胚胎。我们描述了一个管道，用于确定相互作用的平行亲和捕获（iPAC），并显示其使用通过确定合作伙伴的几种蛋白质诱饵的大小和亚细胞位置的范围。该纯化方案平行使用不同的标签，并涉及所得质谱数据集的详细比较，确保所获得的相互作用列表具有高置信度。我们表明，这种方法确定已知的诱饵蛋白的相互作用以及新的相互作用的合作伙伴，通过比较数据与已公布的相互作用数据集。这里提供的体内蛋白质数据集的高置信度为目前不完整的D.黑腹相互作用组此外，我们报告的污染蛋白质是持久的亲和纯化，无论标记的诱饵。
Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.