Wang Linna, Pan Li, Tao W Andy
Department of Biochemistry, ‡Department of Medicinal Chemistry & Molecular Pharmacology, and §Center for Cancer Research, Purdue University , West Lafayette, Indiana 47907, United States.
Anal Chem. 2014 Jul 15;86(14):6741-7. doi: 10.1021/ac501641u. Epub 2014 Jun 30.
The applicability of gel-based proteomic strategies in phosphoproteomics has been largely limited by the lack of technologies for specific detection of phosphoproteins in gels. Here for the first time we report a strategy for simultaneous visualization and identification of phosphoproteome in gels (VIPing) through coupling specific detection of phosphoproteins with protein identification and phosphorylation site mapping by tandem mass spectrometry. The core of the strategy is a novel compound multifunctionalized with a titanium ion(IV) for outstanding selectivity toward phosphorylated residues, a fluorophore for visualization, and a biotin group for phosphopeptide enrichment. The sensitivity and specificity of the VIPing strategy was demonstrated using standard protein mixtures and complex cell extracts, and the method was applied to study the phosphorylation changes of an essential tyrosine kinase Syk and interacting proteins upon B-cell stimulation. The novel technique provides a powerful platform for gel-based phosphoproteomic studies.
基于凝胶的蛋白质组学策略在磷酸化蛋白质组学中的应用在很大程度上受到凝胶中磷酸化蛋白质特异性检测技术缺乏的限制。在此，我们首次报道了一种通过将磷酸化蛋白质的特异性检测与蛋白质鉴定以及串联质谱法对磷酸化位点的定位相结合，在凝胶中同时可视化和鉴定磷酸化蛋白质组（VIPing）的策略。该策略的核心是一种新型化合物，它用四价钛离子进行多功能化，对磷酸化残基具有出色的选择性，带有用于可视化的荧光团和用于磷酸肽富集的生物素基团。使用标准蛋白质混合物和复杂细胞提取物证明了VIPing策略的灵敏度和特异性，并将该方法应用于研究B细胞刺激后关键酪氨酸激酶Syk及其相互作用蛋白的磷酸化变化。这项新技术为基于凝胶的磷酸化蛋白质组学研究提供了一个强大的平台。
