Suzuki–Miyaura cross-coupling for chemoproteomic applications

Abstract

Bioorthogonal chemistry is a mainstay of chemoproteomic sample preparation workflows. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper-catalyzed azide –alkyne cycloaddition (CuAAC) or 'click' chemistry. Here we demonstrate that gel-based activity-based protein profiling (ABPP) and mass-spectrometry-based chemoproteomic profiling can be conducted using Suzuki–Miyaura cross-coupling. We identify reaction conditions that proceed in complex cell lysates and find that Suzuki –Miyaura cross-coupling and CuAAC yield comparable chemoproteomic coverage. Importantly, Suzuki–Miyaura is also compatible with chemoproteomic target deconvolution, as demonstrated using structurally matched probes tailored to react with the cysteine protease caspase-8. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual protein labeling.

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 CHCl3/MeOH protein precipitation, we found that the SP3 method, which relies on carboxyl coated magnetic beads, yields better coverage of biotin-labelled peptides.…”

“…Our recent study revealed that solid phase-enhanced samplepreparation (SP3), 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 µM 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 biotin-azide). 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.…”

“…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 labelled 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 (online-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 phaseenhanced 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 CHCl3/MeOH cleanup, both by improving coverage of labeled peptides and by reducing the amount of protein material required for chemoproteomic studies.…”

SP3-FAIMS Chemoproteomics for High Coverage Profiling of the Human Cysteinome

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