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

Area of Science:

• Chemical proteomics
• Drug discovery
• Mass spectrometry

Background:

• Affinity capture (AC) coupled with mass spectrometry (MS) is crucial for assessing small-molecule target engagement and selectivity in drug discovery.
• Current AC-MS methods face limitations in high-throughput applications due to lengthy sample preparation and MS acquisition.
• There is a need for streamlined, automated workflows to accelerate target engagement studies.

Purpose of the Study:

• To develop and validate an automated, high-throughput AC-MS workflow for small-molecule target profiling.
• To implement a data-independent acquisition (DIA)-MS method for enhanced kinome coverage and quantitative analysis.
• To demonstrate the workflow's utility in assessing drug selectivity against a broad range of kinases.

Main Methods:

• An automated 96-well plate AC workflow using biotinylated probes and streptavidin magnetic beads for target enrichment.
• Direct sampling from EvoSep Solid Phase Extraction tips for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
• Development of a label-free quantitative DIA-MS method for kinome profiling.

Main Results:

• The automated workflow significantly reduced overall and hands-on sample preparation time.
• DIA-MS achieved coverage of approximately 380 kinases, a >60% increase over data-dependent acquisition (DDA)-MS.
• Reproducible target profiling of dasatinib and selectivity assessment of two CDK9 inhibitors against ~250 kinases were demonstrated.

Conclusions:

• The developed automated AC-MS workflow enables efficient and high-throughput target engagement and selectivity profiling.
• This approach significantly improves kinase coverage and reduces analysis time compared to traditional methods.
• The workflow provides a valuable roadmap for accelerating drug discovery and development pipelines.