IDENTIFICATION OF ELECTROPHILICLY MODIFIED PROTEINS IN EUKARYOTIC CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们已经合成了一些亲电的小分子来询问真核细胞的蛋白质组。我们的化合物包含一个半混杂的激酶抑制剂支架，一个末端的炔，和一个反应性的电泳体。这种结合以结合支架并包含形成特定共价键所必需的亲核性的独特的亚类激酶为靶标。结合是通过使用炔和铜催化的“点击化学”来连接不同的荧光和亲和标记来检测的。这些化合物的细胞靶标在处理裂解产物或整个细胞后用质谱仪鉴定，然后亲和纯化共价结合蛋白。标记细胞或复杂混合物中的蛋白质可以揭示我们的抑制剂以及相关分子的生理相关靶点。我们还开发了一种竞争性标记策略，以确定我们的不可逆探针所针对的潜在的非共价激酶抑制剂。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have synthesized a number of electrophilic small molecules to interrogate the proteomes of eukaryotic cells. Our compounds incorporate a semi-promiscuous kinase inhibitor scaffold, a terminal alkyne, and a reactive electrophile. This combination targets unique sub-classes of kinases that both bind the scaffold and contain a requisite nucleophile to form a specific covalent bond. Binding is detected using the alkyne and copper-catalyzed "click chemistry" to attach diverse fluorescent and affinity tags. The compounds' cellular targets are identified by mass spectrometry after treating lysates or whole cells then affinity purifying covalently bound proteins. Labeling of proteins in cells or complex mixtures can reveals the physiologically relevant targets of our inhibitors, as well as related molecules. We have also developed a competitive labeling strategy to identify potential non-covalent inhibitors of kinases targeted by our irreversible probes.