Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)^G12C and Bruton’s tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.

目前用于测量氨基酸侧链反应性的方法缺乏筛选大型化学库以了解蛋白质组相互作用所需的通量。在这里，我们通过使用更小的基于脱硫生物素的探针、样品多路复用、减少的蛋白质起始量和软件来重新设计基于活性的活性半胱氨酸残基蛋白质分析的工作流程，以促进质谱仪上的实时数据采集。我们的方法，简化的基于半胱氨​​酸活性的蛋白质分析 (SLC-ABPP)，实现了样品通量 42 倍的提高，对应于在每个化合物 18 分钟的深度 >8,000 个反应性半胱氨酸位点分析库成员。我们应用它来识别突变体 Kirsten 大鼠肉瘤 (KRAS) ^{G12C的共价抑制剂的蛋白质组范围目标}和布鲁顿氏酪氨酸激酶 (BTK)。此外，我们在三个人类细胞系中创建了对 285 种亲电子试剂具有半胱氨酸反应性的资源，其中包括来自每个细胞系 >6,000 种蛋白质的 >20,000 个半胱氨酸。在多个细胞环境下跨千个成员库对半胱氨酸反应性进行全蛋白质组分析的目标现已触手可及。