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

Abstract

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.

SP3‐FAIMS Chemoproteomics for High‐Coverage Profiling of the Human Cysteinome**

“…As observed both in our prior study [27] and here, sample decontamination for chemoproteomic applications that rely on increased concentration of biotin capture reagents (e. g., biotinazide and related molecules) is an essential step to achieve high coverage of biotinylated peptides. When benchmarked against standard CHCl 3 /MeOH protein precipitation, we found that the SP3 method yields better coverage of biotin-labeled peptides.…”

“…Our recent study revealed that SP3 technology, a method that uses carboxyl coated magnetic beads to separate proteins and peptides from contaminates, can increase the recovery of biotinylated peptides when lower concentrations of IAA and biotin-azide were used for labeling (200 and 400 μM, respectively). [27] Therefore, we sought to determine whether this improved recovery would extend to samples treated with our optimized labeling conditions (2 mM IAA and 4 mM biotinazide). Our first step was to optimize two key parameters for SP3 cleanup, 1) the ratio of protein material to magnetic beads and 2) elution conditions to maximize recovery of peptides after cleanup.…”

“…Synthesis of reagents. Compound 1 and 3 were prepared as has been reported [27] and synthesis of compound 4 was performed according to the previous studies. [12]…”

“…Additionally, while CuAAC is the biorthogonal reaction of choice for chemoproteomics, our recent study revealed incomplete biotinylation as a potential confounder to achieving high coverage of labeled peptides. [27] An ideal chemoproteomics experiment would identify nearly all cysteine-containing peptides with minimal hours of instrument time. Achieving this ideal will require near complete biotinylation of all cysteines, efficient enrichment and release from avidin resin, an optimized liquid chromatography gradient together with fractionation (on-line or off-line).…”

“…Two key recent technical innovations should enable such comprehensive chemoproteomic coverage. First, our recent work [27] revealed that, when applied to chemoproteomics, the single-pot, solid phase-enhanced sample-preparation (SP3) protein extraction technique [28][29][30] enabled the rapid removal of contaminants, including biotin reagents. We found that SP3 compares favorably to chloroform/methanol (CHCl 3 /MeOH) cleanup, both by improving coverage of labeled peptides and by reducing the amount of protein material required for chemoproteomic studies.…”