Novel mass spectrometry-based assay for thymidylate synthase activity

Thymidylate synthase (TS) is an enzyme responsible for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), with the co-substrate 5,10-methylenetetrahydrofolate (5,10-CH₂-THF) as the methyl donor. TS is the only enzyme capable of de novo biosynthesis of dTMP in humans, a nucleotide crucial for DNA synthesis and therefore cell proliferation and survival. As such, TS is a major drug target in chemotherapy by compounds such as 5-fluorouracil. Due to the clinical and physiological importance of TS, the ability to accurately assay its activity is crucial. Several assays have been developed for this purpose, relying on spectrophotometry or radioisotope labeling methods. In this study, we have developed a liquid chromatography - mass spectrometry-based method for assessing TS activity by direct and specific measurement of the reaction product, dTMP. The assay was tested on mouse liver homogenates. We noted that excessive 5,10-CH₂-THF concentration (400 µM) led to substrate inhibition and therefore 200 µM was used. The activity assayed at 1 µM dUMP was linear with protein content and time (up to 60 min) and was 0.56 ± 0.12 pmol/mg protein/min, in line with previously reported values. Additionally, by using a high mass resolution Orbitrap instrument side reactions were monitored, revealing major changes in folate pools and nucleotide metabolism. These findings highlight the value of the developed TS assay for routine TS activity monitoring in complex matrixes such as clinical material.