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.

化学蛋白质组学是一种强大的方法，能够检测小分子和蛋白质组之间的相互作用；然而，迄今为止，其作为化学文库高通量筛选方法的应用受到限制。为了满足这一需求，我们进一步开发了化学蛋白质组学工作流程，使用基于活性的蛋白质分析筛选细胞裂解物中针对去泛素化 (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，从而能够在 96 孔板格式中进行靶标识别和共价片段库分析。使用数据独立采集 (DIA) 方法进行质谱 (MS) 分析，并结合自动 Evosep 液相色谱 (LC)，将每个样品的仪器运行时间缩短至 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 种 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测定验证了针对 OTUD7B 和 UCHL3 的 4 个命中片段。