Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.
Curr Protoc. 2022 Jul;2(7):e490. doi: 10.1002/cpz1.490.
The proteasome is a multisubunit protein complex responsible for the degradation of proteins, making it essential in myriad cellular processes. Several reversible and irreversible peptide substrates inspired by known proteasome inhibitors have been developed to visualize it and monitor its activity; however, they have limited commercial availability or possess fluorophores that overlap with other known chemical probes, limiting their simultaneous use. The protocols presented here describe the synthesis of a clickable epoxomicin-based probe followed by the copper-catalyzed installment of an azide-containing fluorophore, and the application of the synthesized peptide in proteasome activity assays by SDS-PAGE and flow cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of clickable peptide fragment (2) Basic Protocol 2: In-solution coupling of epoxy-ketone moiety to fragment (2) Basic Protocol 3: Copper-catalyzed click reaction of (3) with fluorophore of choice Basic Protocol 4: Monitoring proteasome activity by SDS-PAGE in HEK-293T cells Alternate Protocol: Monitoring proteasome activity by flow cytometry in HEK-293T cells.
蛋白酶体是一种多亚基蛋白复合物，负责蛋白质的降解，在众多细胞过程中是必不可少的。已经开发了几种基于已知蛋白酶体抑制剂的可逆和不可逆肽底物来可视化和监测其活性；然而，它们的商业可用性有限，或者具有与其他已知化学探针重叠的荧光团，限制了它们的同时使用。这里介绍的方案描述了一种点击型环氧酮类蛋白酶体探针的合成，然后是含有叠氮化物的荧光团的铜催化安装，以及合成肽在 SDS-PAGE 和流式细胞术测定蛋白酶体活性中的应用。© 2022 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：点击肽片段（2）的固相合成 基本方案 2：将环氧酮部分在溶液中偶联到片段（2）上 基本方案 3：（3）与所选荧光团的铜催化点击反应 基本方案 4：用 SDS-PAGE 在 HEK-293T 细胞中监测蛋白酶体活性 备选方案：用流式细胞术在 HEK-293T 细胞中监测蛋白酶体活性。
