Schroeder Melanie J, Webb Donna J, Shabanowitz Jeffrey, Horwitz Alan F, Hunt Donald F
Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22904, USA.
J Proteome Res. 2005 Sep-Oct;4(5):1832-41. doi: 10.1021/pr0502020.
Methods for the simultaneous identification of interacting proteins and post-translational modifications of the focal adhesion adapter protein, paxillin, are presented. The strategy includes (1) lower-level, transient transfection of FLAG-GFP-Paxillin into HEK293 cells, (2) incubation of cells with phosphatase inhibitors prior to lysis, (3) purification of paxillin by anti-FLAG immunoprecipitation, (4) analysis of peptides generated from on-beads digestion using LTQ-FT or LTQ-ETD mass spectrometry, and (5) enrichment of phosphopeptide methyl esters with IMAC. Using the above strategies, we identify 29 phosphorylation sites (19 novel and 10 previously reported) and a novel glycosylation site on Ser 74. Furthermore, with this method, we simultaneously detect 10 co-purifying proteins which are present in focal adhesion complexes. Extension of these methods to other substrates should facilitate generation of global phosphorylation maps and protein-protein interactions for any protein of interest.
本文介绍了同时鉴定粘着斑衔接蛋白桩蛋白的相互作用蛋白和翻译后修饰的方法。该策略包括：（1）将FLAG-GFP-桩蛋白进行低水平瞬时转染至HEK293细胞；（2）在裂解前用磷酸酶抑制剂孵育细胞；（3）通过抗FLAG免疫沉淀法纯化桩蛋白；（4）使用LTQ-FT或LTQ-ETD质谱分析珠上消化产生的肽段；（5）用IMAC富集磷酸肽甲酯。使用上述策略，我们鉴定出29个磷酸化位点（19个新位点和10个先前报道的位点）以及Ser 74上一个新的糖基化位点。此外，用这种方法，我们同时检测到10个共纯化蛋白，它们存在于粘着斑复合物中。将这些方法扩展到其他底物应有助于生成任何感兴趣蛋白质的全局磷酸化图谱和蛋白质-蛋白质相互作用。
