Multiplexed CuAAC Suzuki–Miyaura Labeling for Tandem Activity-Based Chemoproteomic Profiling

Mass-spectrometry-based
chemoproteomics has enabled the rapid and
proteome-wide discovery of functional and potentially ’druggable’
hotspots in proteins. While numerous transformations are now available,
chemoproteomic studies still rely overwhelmingly on copper­(I)-catalyzed
azide–alkyne cycloaddition (CuAAC) or ’click’
chemistry. The absence of bio-orthogonal chemistries that are functionally
equivalent and complementary to CuAAC for chemoproteomic applications
has hindered the development of multiplexed chemoproteomic platforms
capable of assaying multiple amino acid side chains in parallel. Here,
we identify and optimize Suzuki–Miyaura cross-coupling conditions
for activity-based protein profiling and mass-spectrometry-based chemoproteomics,
including for target deconvolution and labeling site identification.
Uniquely enabled by the observed orthogonality of palladium-catalyzed
cross-coupling and CuAAC, we combine both reactions to achieve dual
labeling. Multiplexed targeted deconvolution identified the protein
targets of bifunctional cysteine- and lysine-reactive probes.

基于质谱的化学蛋白质组学技术已使得蛋白质中功能性和潜在的‘可药化’热点区域得以快速且全蛋白质组层面的发现。尽管目前已有多种转化技术可用，化学蛋白质组学研究仍绝大多数依赖于铜(I)催化的叠氮化物-炔烃环加成反应（CuAAC）或所谓的‘点击’化学。由于缺乏与CuAAC在功能上等效且互补的生物正交化学，这阻碍了能够并行检测多种氨基酸侧链的多重化学蛋白质组平台的开发。在此，我们鉴定并优化了Suzuki-Miyaura交叉偶联条件，以用于基于活性的蛋白质分析和质谱化学蛋白质组学，包括针对目标解析和标记位点识别。得益于观察到的钯催化交叉偶联与CuAAC的互斥性，我们结合了这两种反应以实现双重标记。多重靶向解析识别了双功能半胱氨酸和赖氨酸反应探针的蛋白质靶点。