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.