Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.
Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.
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DOI：
10.1021/bi201636s
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发表时间：
2011-12-27
期刊：
影响因子：
2.9
通讯作者：
Best, Michael D.
中科院分区：
文献类型：
--
作者：
Rowland, Meng M.;Bostic, Heidi E.;Gong, Denghuang;Speers, Anna E.;Lucas, Nathan;Cho, Wonhwa;Cravatt, Benjamin F.;Best, Michael D.
磷脂酰肌醇多磷脂，如磷脂酰肌醇(3，4，5)-三磷酸(PI(3，4，5)P3)，调节着重要的生物学过程，其中许多在疾病中是异常的。在加强蛋白质结合伙伴的膜结合的相互作用中，这些脂类通常作为特定部位的配体。在这里，我们描述了与PI(3，4，5)P3头部基团相对应的双功能活性探针的发展，这些探针可以有效地从复杂的样本中识别和表征蛋白质结合伙伴，即癌细胞提取物。这些探针既包含用于目标蛋白质共价标记的光亲和标签，也包含用于随后检测或操纵标记蛋白质的二级手柄。利用带有不同二级标记的探针，或者通过直接连接用于光学检测的荧光染料，或者通过使用可在蛋白质标记后通过点击化学进行衍生化的炔烃。首先，我们描述了用于产生具有不同报告标签的多个探针的设计和模块化合成策略，用于表征探针标记的蛋白质。接下来，我们报道了使用纯化的蛋白，Akt的PH结构域进行的初步标记研究，其中发现了标记该靶标的探针，通过凝胶上检测判断。此外，蛋白质标记被对照取消，包括与未标记的PI(3，4，5)P3头基类似物的竞争以及通过蛋白质变性，表明特异性标记。此外，PI(3，4，5)P3头基和光亲和标签之间具有不同接头长度的探针导致了蛋白质标记的变化，表明在这种情况下较短的接头更有效。最后，利用细胞提取液进行蛋白质组学标记研究，通过凝胶内检测观察标记的蛋白质，并用生物素后标记、亲和层析和串联质谱仪鉴定标记的蛋白质。这些研究共产生了265个蛋白质，包括已知和新的候选PI(3，4，5)P3结合蛋白。
Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane-association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins as well as a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by on-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analog as well as through protein denaturation, indicating specific labeling. In addition, probes featuring different linker lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts, labeled proteins were observed by in-gel detection and characterized using post-labeling with biotin, affinity chromatography and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P3-binding proteins.
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