Shikwana Flowreen, Heydari Beeta S, Ofori Samuel, Truong Cindy, Turmon Alexandra C, Darrouj Joelle, Holoidovsky Lara, Gustafson Jeffrey L, Backus Keriann M
Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA; Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, 90095, USA.
Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA. 92182, USA.
Mol Cell Proteomics. 2024 Dec 18:100898. doi: 10.1016/j.mcpro.2024.100898.
Cysteine chemoproteomic screening platforms are widely utilized for chemical probe and drug discovery campaigns. Chemoproteomic compound screens, which use a mass spectrometry-based proteomic readout, can interrogate the structure activity relationship (SAR) for thousands of proteins in parallel across the proteome. The versatility of chemoproteomic screens has been demonstrated across electrophilic, nucleophilic, and reversible classes of molecules. However, a key bottleneck that remains for these approaches is the low throughput nature of most established sample preparation workflows, which rely on many time-intensive and often error prone steps. Addressing these challenges, here we establish a novel workflow, termed CySP3-96, that pairs single-pot, solid-phase-enhanced, sample preparation (SP3) with a customized 96-well sample cleanup workflow to achieve streamlined multiplexed sample preparation. Our CySP3-96 method addresses prior volume limitations of SP3, which allows for seamless 96-well chemoproteomic sample preparation, including for large input amounts that are incompatible with prior methods. By deploying CySP3-96 to screen a focused set of 16 cysteine-reactive compounds, we identify 2633 total ligandable cysteines, including 21 not captured in CysDB. Chemoproteomic analysis of a pair of atropisomeric electrophilic kinase inhibitors reveals striking stereoselective cysteine ligandability for 67 targets across the proteome. When paired with our innovative budget friendly magnetic resin, CySP3-96 represents a versatile, low cost, and highly reproducible screening platform with widespread applications spanning all types of chemoproteomic studies.
半胱氨酸化学蛋白质组学筛选平台被广泛用于化学探针和药物发现研究。化学蛋白质组学化合物筛选使用基于质谱的蛋白质组学读数，可以在整个蛋白质组中并行研究数千种蛋白质的结构活性关系（SAR）。化学蛋白质组学筛选的多功能性已在亲电、亲核和可逆分子类别中得到证明。然而，这些方法仍然存在的一个关键瓶颈是大多数既定样品制备工作流程的低通量性质，这些流程依赖于许多耗时且容易出错的步骤。为应对这些挑战，我们在此建立了一种名为CySP3-96的新型工作流程，该流程将单锅、固相增强样品制备（SP3）与定制的96孔样品净化工作流程相结合，以实现简化的多重样品制备。我们的CySP3-96方法解决了SP3先前的体积限制问题，从而能够无缝进行96孔化学蛋白质组学样品制备，包括处理与先前方法不兼容的大量输入样品。通过部署CySP3-96来筛选一组聚焦的16种半胱氨酸反应性化合物，我们总共鉴定出2633个可配体半胱氨酸，其中包括21个未在CysDB中捕获的半胱氨酸。对一对阻转异构亲电激酶抑制剂进行化学蛋白质组学分析，揭示了整个蛋白质组中67个靶点具有显著的立体选择性半胱氨酸配体结合能力。当与我们创新的经济实惠的磁性树脂相结合时，CySP3-96代表了一个多功能、低成本且高度可重复的筛选平台，广泛应用于所有类型的化学蛋白质组学研究。
