Automated High-Throughput Affinity Capture-Mass Spectrometry Platform with Data-Independent Acquisition

Affinity capture (AC) combined with
mass spectrometry
(MS)-based
proteomics is highly utilized throughout the drug discovery pipeline
to determine small-molecule target selectivity and engagement. However,
the tedious sample preparation steps and time-consuming MS acquisition
process have limited its use in a high-throughput format. Here, we
report an automated workflow employing biotinylated probes and streptavidin
magnetic beads for small-molecule target enrichment in the 96-well
plate format, ending with direct sampling from EvoSep Solid Phase
Extraction tips for liquid chromatography (LC)-tandem mass spectrometry
(MS/MS) analysis. The streamlined process significantly reduced both
the overall and hands-on time needed for sample preparation. Additionally,
we developed a data-independent acquisition-mass spectrometry (DIA-MS)
method to establish an efficient label-free quantitative chemical
proteomic kinome profiling workflow. DIA-MS yielded a coverage of
∼380 kinases, a > 60% increase compared to using a data-dependent
acquisition (DDA)-MS method, and provided reproducible target profiling
of the kinase inhibitor dasatinib. We further showcased the applicability
of this AC-MS workflow for assessing the selectivity of two clinical-stage
CDK9 inhibitors against ∼250 probe-enriched kinases. Our study
here provides a roadmap for efficient target engagement and selectivity
profiling in native cell or tissue lysates using AC-MS.