Maccarrone Giuseppina, Bonfiglio Juan Jose, Silberstein Susana, Turck Christoph W, Martins-de-Souza Daniel
Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich, Germany.
Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET, Partner Institute of the Max Planck Society, Buenos Aires, Argentina.
Methods Mol Biol. 2017;1546:223-234. doi: 10.1007/978-1-4939-6730-8_19.
Identifying the partners of a given protein (the interactome) may provide leads about the protein's function and the molecular mechanisms in which it is involved. One of the alternative strategies used to characterize protein interactomes consists of co-immunoprecipitation (co-IP) followed by shotgun mass spectrometry. This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions. In this chapter, we describe a co-IP protocol for interactome studies that uses an antibody against a protein of interest bound to protein A/G plus agarose beads to isolate a protein complex. The interacting proteins may be further fractionated by SDS-PAGE, followed by in-gel tryptic digestion and nano liquid chromatography high-resolution tandem mass spectrometry (nLC ESI-MS/MS) for identification purposes. The computational tools, strategy for protein identification, and use of interactome databases also will be described.
确定某一特定蛋白质的相互作用伙伴（即相互作用组），可能会为该蛋白质的功能以及它所涉及的分子机制提供线索。用于表征蛋白质相互作用组的替代策略之一，是先进行免疫共沉淀（co-IP），然后进行鸟枪法质谱分析。这能够在生理条件下，从细胞或组织裂解物中分离并鉴定处于天然状态的蛋白质靶点及其相互作用组。在本章中，我们描述了一种用于相互作用组研究的免疫共沉淀方案，该方案使用与蛋白A/G加琼脂糖珠结合的针对目标蛋白的抗体来分离蛋白质复合物。相互作用的蛋白质可通过SDS-PAGE进一步分级分离，随后进行胶内胰蛋白酶消化和纳升液相色谱高分辨率串联质谱分析（nLC ESI-MS/MS）以进行鉴定。还将介绍计算工具、蛋白质鉴定策略以及相互作用组数据库的使用。
