Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Drug Discovery

Background:

• Label-free mass spectrometry (MS) is crucial for enzyme assay development in drug discovery.
• MS allows sensitive detection of enzyme substrates/products in biological samples without labeling.
• Cardiovascular drug discovery requires efficient screening for enzyme modulators.

Purpose of the Study:

• To develop a high-throughput screening (HTS) assay for cystathionine beta-synthase (CBS) activity using label-free MS.
• To quantify the unlabeled product, cystathionine, using RapidFire((R)) high-throughput MS (HTMS).
• To assess the utility of the HTMS assay in complex biological matrices for secondary screening.

Main Methods:

• Developed an in vitro HTS assay for CBS activity utilizing label-free HTMS.
• Quantified cystathionine with a 7-second sample throughput and a linear range of 80-10,000 nM.
• Screened a library of 25,559 compounds in 8 days.

Main Results:

• Achieved a hit rate of 0.32% with a 90% confirmation rate for active compounds.
• Demonstrated successful application of the HTMS assay to secondary screens in serum, cell-based assays, and liver explants without modification.
• The assay exhibited high throughput and reliability for large-scale screening.

Conclusions:

• Label-free HTMS is a powerful and versatile tool for enzyme activity screening in drug discovery.
• The developed CBS assay is efficient, accurate, and adaptable to various biological matrices.
• This approach significantly reduces development time for drug discovery projects.