Accelerating multiplexed profiling of protein-ligand interactions: high-throughput plate-based reactive cysteine profiling with minimal input

Chemoproteomics investigates small molecule-protein interactions and has made significant progress in recent years. Despite its vast potential, the proteome-wide profiling of reactive cysteine ligandability remains a formidable task to adapt for high throughput applications. This is primarily due to a lack of platforms capable of achieving the desired depth using low sample input in 96- or 384-well plates. Here we have revamped the cysteine profiling platform to address the challenge with an eye toward performing high-throughput library screening in plates. By incorporating several changes including i) an 18-plex TMT sample multiplexing strategy, ii) a magnetic beads-based one-pot workflow, iii) a 10X higher capacity streptavidin resin, and iv) optimized mass spectrometry analyses, a plate-based platform was developed that enables routine interrogation of either ~18,000 or ~24,000 reactive cysteines based on starting amounts of 10 or 20 µg, respectively. We applied the platform to screen a library of 192 electrophiles in the native HEK293T proteome, mapping the ligandablity of 38,450 reactive cysteines from 8,274 human proteins. The significantly improved depth revealed many previously unknown reactive cysteines and cysteine-ligand interactions and led to the identification of an azepane-containing acrylamide which has preferential binding to cysteines in EGF-like domains. We further applied the platform to characterize new cellular targets of well-studied compounds and covalent drugs in three different human cell lines. We found that ARS-1620, a KRASG12C inhibitor, also binds to cysteine 140 of an off-target adenosine kinase ADK, inhibiting its kinase activity. The platform represents a major step forward to high throughput evaluation of reactive cysteines on a proteome-wide scale.化学蛋白质组学（Chemoproteomics）专注于研究小分子与蛋白质的相互作用，近年来取得了显著进展。尽管该领域潜力巨大，但针对反应性半胱氨酸配基结合性的全蛋白质组范围分析，仍是适配高通量应用场景的一项艰巨挑战。这一困境的主要成因在于，目前缺乏能够在96孔或384孔板中以低样本投入量实现所需覆盖深度的分析平台。本研究针对该难题对半胱氨酸谱分析平台进行了优化升级，旨在实现基于微孔板的高通量文库筛选。通过整合多项改进措施：①18重TMT（串联质量标签，Tandem Mass Tag）样本多重标记策略、②基于磁珠的一锅法操作流程、③10倍负载容量的链霉亲和素树脂，以及④优化的质谱分析流程，最终开发出一款基于微孔板的分析平台。该平台可根据起始样本量分别为10 μg或20 μg时，常规检测约18000个或约24000个反应性半胱氨酸位点。本研究将该平台应用于在天然HEK293T蛋白质组中筛选192种亲电试剂文库，成功绘制了来自8274个人类蛋白质中38450个反应性半胱氨酸位点的配基结合性谱图。该平台大幅提升的覆盖深度揭示了大量此前未被报道的反应性半胱氨酸位点及半胱氨酸-配基相互作用，并鉴定出一种含氮杂环庚烷的丙烯酰胺类化合物，该化合物可优先结合表皮生长因子样结构域中的半胱氨酸残基。此外，本研究进一步将该平台应用于三种不同人类细胞系中，对经典研究化合物及共价药物的新型细胞靶点进行了表征。研究发现，KRASG12C抑制剂ARS-1620还可结合脱靶靶点腺苷激酶ADK的第140位半胱氨酸残基，从而抑制其激酶活性。该平台代表了全蛋白质组范围内反应性半胱氨酸高通量评估领域的重要进展。