CySP3-96 enables scalable, streamlined, and low-cost sample preparation for cysteine chemoproteomic applications.

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.