Novel mass spectrometry-based assay for thymidylate synthase activity

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Thymidylate synthase (TS) is vital for DNA synthesis and a key target in cancer chemotherapy.
• Existing methods for assaying TS activity have limitations.
• Accurate TS activity measurement is crucial for clinical and research applications.

Purpose of the Study:

• To develop and validate a novel liquid chromatography-mass spectrometry (LC-MS) based assay for thymidylate synthase (TS) activity.
• To enable direct and specific measurement of the TS reaction product, deoxythymidine monophosphate (dTMP).
• To assess the utility of the developed assay in complex biological matrices.

Main Methods:

• Development of an LC-MS method for quantifying dTMP, the product of TS enzymatic activity.
• Testing the assay using mouse liver homogenates.
• Optimization of co-substrate (5,10-methylenetetrahydrofolate) concentration to avoid substrate inhibition.
• Monitoring of side reactions and folate pool changes using high-mass-resolution Orbitrap instrumentation.

Main Results:

• The developed LC-MS assay provides a direct and specific measurement of TS activity.
• TS activity was found to be linear with protein content and incubation time (up to 60 minutes) at optimized substrate concentrations.
• Assayed TS activity was 0.56 ± 0.12 pmol/mg protein/min, consistent with literature values.
• The assay revealed significant alterations in folate pools and nucleotide metabolism.

Conclusions:

• The novel LC-MS assay is a robust and accurate method for determining TS activity.
• The assay is suitable for routine monitoring of TS activity in complex samples, including clinical materials.
• This method offers advantages over traditional spectrophotometric and radioisotope-based assays.