A novel proteomic screen for peptide-protein interactions
A novel proteomic screen for peptide-protein interactions
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DOI：
10.1074/jbc.m309909200
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发表时间：
2004-03-12
影响因子：
4.8
通讯作者：
Mann, M
中科院分区：
文献类型：
--
作者：
Schulze, WX;Mann, M
短的、非结构化的氨基酸序列和模块化蛋白结构域之间的调控相互作用是细胞信号传递的中心。在这里，我们使用“活性”(例如，磷酸化)和“对照”(例如，非磷酸化)形式的合成肽作为亲和力下拉实验的诱饵，通过定量蛋白质组学来确定这种相互作用。细胞培养中氨基酸的稳定同位素标记直接通过质谱仪(Blagoev，B.，Kratchmarova，I.，Ong，S.-E.，Nielsen，M.，Foster，L.J.和Mann，M.(2003)NAT)测定的同位素比率来区分特定的结合剂。生物技术。21、315-318)。表皮生长因子受体的酪氨酸磷酸化肽特异性地检索到含有Src同源结构域(SH)2-和SH3结构域的适配蛋白Grb2。Seven less的Son的一个富含Pro的序列也特异性地结合了Grb2，表明该屏幕与低亲和力的相互作用保持了特异性。富含脯氨酸的SOS多肽仅检索到含有SH3结构域的蛋白质作为特异性结合伙伴。其中两个为Pacsin 3和Sorting Nexin 9，经免疫沉淀证实。我们的数据与SOS的作用变化是一致的，从依赖RAS的信号到通过Pro-SH3结构域切换的肌动蛋白重塑/内吞信号事件。
Regulated interactions between short, unstructured amino acid sequences and modular protein domains are central to cell signaling. Here we use synthetic peptides in "active" (e.g. phosphorylated) and "control" (e.g. nonphosphorylated) forms as baits in affinity pull-down experiments to determine such interactions by quantitative proteomics. Stable isotope labeling by amino acids in cell culture distinguishes specific binders directly by the isotope ratios determined by mass spectrometry (Blagoev, B., Kratchmarova, I., Ong, S.-E., Nielsen, M., Foster, L. J., and Mann, M. ( 2003) Nat. Biotechnol. 21, 315 - 318). A tyrosine-phosphorylated peptide of the epidermal growth factor receptor specifically retrieved the Src homology domain (SH) 2- and SH3 domain-containing adapter protein Grb2. A proline-rich sequence of Son of Sevenless also specifically bound Grb2, demonstrating that the screen maintains specificity with low affinity interactions. The proline-rich Sos peptide retrieved only SH3 domain containing proteins as specific binding partners. Two of these, Pacsin 3 and Sorting Nexin 9, were confirmed by immunoprecipitation. Our data are consistent with a change in the role of Sos from Ras-dependent signaling to actin remodeling/endocytic signaling events by a proline-SH3 domain switch.