High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry.

Area of Science:

• Analytical Chemistry
• Drug Discovery
• Biochemistry

Background:

• Covalent drugs offer a promising strategy for targeting previously undruggable proteins.
• Traditional mass spectrometry methods (e.g., LC-MS, SPE-MS) are bottlenecks for high-throughput screening of covalent modifiers due to limitations in speed and sample preparation.
• There is a need for faster, more efficient platforms to screen large compound libraries for covalent binders.

Purpose of the Study:

• To introduce and evaluate a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for high-throughput screening of covalent modifiers.
• To assess the feasibility of AEMS for analyzing complex samples with minimal preparation.
• To compare the performance of AEMS with conventional mass spectrometry techniques for covalent modifier screening.

Main Methods:

• Development of a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system.
• Screening of a library of approximately 10,000 covalent modifiers against a 50 kDa target protein (Werner syndrome helicase) in a 1536-well format.
• Direct analysis of samples in high-salt buffer without cleanup, followed by automated data analysis for quantitation of covalent protein-ligand adducts.

Main Results:

• The AEMS system completed the screening of ~10,000 compounds in 17 hours.
• The screening demonstrated high fidelity with a Z' factor of 0.8 and a hit rate of 2.3%.
• 75% of identified hits were confirmed as functional antagonists in subsequent biochemical assays, with AEMS showing low false positive/negative rates compared to LC-MS.

Conclusions:

• The prototype AEMS platform enables robust, high-throughput screening of covalent modifiers.
• AEMS significantly increases throughput (10-100 fold) and library size capacity compared to existing methods, while minimizing sample preparation.
• This innovative approach accelerates the identification of functional covalent drug candidates for challenging therapeutic targets.