IDENTIFICATION OF ELECTROPHILICLY MODIFIED PROTEINS IN EUKARYOTIC CELLS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们已经合成了一些亲电小分子来询问真核细胞的蛋白质组。 我们的化合物包含半混杂激酶抑制剂支架、末端炔和反应性亲电试剂。 这种组合靶向独特的激酶亚类，这些激酶既结合支架又含有必要的亲核试剂以形成特异性共价键。 使用炔和铜催化的“点击化学”来检测结合，以附着不同的荧光和亲和标签。 在处理裂解物或全细胞后，通过质谱法鉴定化合物的细胞靶点，然后亲和纯化共价结合的蛋白质。 细胞或复杂混合物中蛋白质的标记可以揭示我们抑制剂的生理相关靶标以及相关分子。 我们还开发了一种竞争性标记策略，以识别我们的不可逆探针靶向的激酶的潜在非共价抑制剂。

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.