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，已实现蛋白质功能性和潜在“可药物化”热点区域的快速及蛋白质组广域发现。尽管如今已有众多转化技术可供选择，化学蛋白质组学研究依旧主要依赖于铜(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功能等效且互补的生物正交化学，阻碍了多路复用化学蛋白质组平台的开发，这些平台能够并行检测多种氨基酸侧链。在本研究中，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基于质谱的化学蛋白质组学，包括目标去卷积和标记位点识别。得益于观察到的钯催化的交叉偶联与CuAAC的相互正交性，我们将两种反应结合，实现双重标记。多路复用靶向去卷积识别了双功能半胱氨酸和赖氨酸反应探针的蛋白质靶点。