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

Jing, H.; Richardson, P. L.; Potts, G. K.; Senaweera, S.; Marin, V. L.; McClure, R. A.; Banlasan, A.; Tang, H.; Kath, J.; Ma, R.; Williams, J.

doi:10.1101/2024.08.13.607785

bioRxiv · 10.1101/2024.08.13.607785

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

Jing, H.·Richardson, P. L.·Potts, G. K.·Senaweera, S.·Marin, V. L.·McClure, R. A.·Banlasan, A.·Tang, H.·Kath, J.·Ma, R.·Williams, J.

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Abstract

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 has limited its use in high-throughput format. Here, we report an automated workflow employing biotinylated probes and streptavidin magnetic beads for small molecule target enrichment in 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 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 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.

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Jing, H., Richardson, P. L., Potts, G. K., Senaweera, S., Marin, V. L., McClure, R. A., Banlasan, A., Tang, H., Kath, J., Ma, R., Williams, J.. 2024-08-13. An Automated High-throughput Affinity Capture-Mass Spectrometry Platform with Data-Independent Acquisition. https://doi.org/10.1101/2024.08.13.607785

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