Ong Shao-En, Schenone Monica, Margolin Adam A, Li Xiaoyu, Do Kathy, Doud Mary K, Mani D R, Kuai Letian, Wang Xiang, Wood John L, Tolliday Nicola J, Koehler Angela N, Marcaurelle Lisa A, Golub Todd R, Gould Robert J, Schreiber Stuart L, Carr Steven A
Proteomics Platform, The Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA.
Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4617-22. doi: 10.1073/pnas.0900191106. Epub 2009 Mar 2.
Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or target-based screens, is extremely rare. Such a capability is expected to be highly illuminating--providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.
大多数小分子探针和药物通过与一种或多种蛋白质相互作用来改变细胞回路。无论是通过基于细胞的表型筛选还是基于靶点的筛选发现的化合物，对相互作用的蛋白质及其相关蛋白质复合物有全面的了解都极为罕见。这样的能力有望具有高度启发性——为小分子实现其已知作用的机制提供有力线索，并提示潜在的未知作用。我们描述了一种将定量蛋白质组学（SILAC）与亲和富集相结合的强大方法，以无偏向、可靠且全面地鉴定与小分子探针和药物结合的蛋白质。该方法具有可扩展性且通用性强，在不同化合物类别之间几乎无需优化，并且已经对我们对小分子探针的研究产生了变革性影响。在此，我们详细描述该方法在鉴定激酶抑制剂和免疫亲和素结合剂靶点方面的应用。
