A chemoproteomic platform for selective deubiquitinase inhibitor discovery
A chemoproteomic platform for selective deubiquitinase inhibitor discovery
复制标题
DOI：
10.1016/j.xcrp.2023.101636
复制
发表时间：
2023-10-18
影响因子：
8.9
通讯作者：
Bush，Jacob T.
中科院分区：
文献类型：
--
作者：
Cookson，Rosa;Vuorinen，Aini;Bush，Jacob T.
化学蛋白质组学是一种能够检测小分子与蛋白质组相互作用的有效方法，然而，它作为高通量筛选化学文库的方法到目前为止一直受到限制。为了满足这一需求，我们进一步开发了一种化学蛋白质组学工作流程，使用基于活性的蛋白质图谱来筛选细胞裂解产物中针对去泛素化(DUB)酶的半胱氨酸反应性共价片段。通过使用靶向泛素探针，我们解决了灵敏度和亲和力限制，使目标识别和96孔板格式的共价片段文库分析成为可能。将数据独立采集(DIA)方法用于质谱仪(MS)分析，并结合自动Evosep液相色谱(LC)，将仪器运行时间减少到每个样品21分钟，并简化了工作流程。在这项概念验证研究中，我们分析了针对57个DUB蛋白的138个共价片段，并通过位点鉴定实验和正交生化活性分析验证了四个针对OTUD7B和UCHL3的HIT片段。
Chemoproteomics is a powerful method capable of detecting interactions between small molecules and the proteome; however, its use as a high-throughput screening method for chemical libraries has so far been limited. To address this need, we have further developed a chemoproteomics workflow to screen cysteine-reactive covalent fragments in cell lysates against the deubiquitinating (DUB) enzymes using activity-based protein profiling. By using targeted ubiquitin probes, we have addressed sensitivity and affinity limitations, enabling target identification and covalent fragment library profiling in a 96-well plate format. The use of data-independent acquisition (DIA) methods for mass spectrometry (MS) analysis combined with automated Evosep liquid chromatography (LC) reduced instrument runtimes to 21 min per sample and simplified the workflow. In this proof-of-concept study, we profile 138 covalent fragments against 57 DUB proteins and validate four hit fragments against OTUD7B and UCHL3 through site identification experiments and orthogonal biochemical activity assays.