Institute of Chemical Sciences and Engineering, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
J Am Chem Soc. 2009 Dec 16;131(49):17954-62. doi: 10.1021/ja907818q.
We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence scanning. S-CROSS requires the expression of the putative interacting proteins as fusion to CLIP-tag or SNAP-tag, two protein tags that can be specifically labeled with synthetic probes. Bifunctional molecules that contain the substrates of the two tags connected via a fluorophore are used to selectively cross-link interacting proteins in cell lysate. The amount of trapped complex can be then quantified after SDS gel electrophoresis by in-gel fluorescence scanning. On the basis of a detailed kinetic analysis of the cross-linking reaction, we showed that the cross-linking efficiency can be used as an indicator of interaction between two proteins, allowing thereby the unambiguous identification of interacting protein pairs. We validated our approach by confirming a number of interactions through selective cross-linking and showed that it permits the quantitative and simultaneous analysis of multiple homotypic and heterotypic protein complexes and the differentiation between strong and weak protein-protein interactions.
我们设计了一些分子，可在细胞裂解物中实现相互作用蛋白的选择性交联（S-CROSS），并通过胶内荧光扫描对捕获的复合物进行灵敏检测。S-CROSS 需要将假定的相互作用蛋白表达为 CLIP 标签或 SNAP 标签融合蛋白，这两种蛋白标签可以用合成探针特异性标记。包含两个标签的底物通过荧光团连接的双功能分子用于选择性地交联细胞裂解物中的相互作用蛋白。然后，通过 SDS 凝胶电泳后的胶内荧光扫描定量捕获的复合物量。基于对交联反应的详细动力学分析，我们表明交联效率可作为两种蛋白质之间相互作用的指标，从而可以明确鉴定相互作用的蛋白质对。我们通过选择性交联验证了我们的方法，并证实了许多相互作用，表明它允许对多种同型和异型蛋白复合物进行定量和同时分析，并区分强和弱的蛋白-蛋白相互作用。
