Comprehensive analysis of sulfotransferase activity using LC-MS/MS

This article is based on a poster originally authored by Jing Lai, Gang Yang, Yushi Cao, Xuehua Zhang, Yi Li, Bojia Liu, Youpeng Yang, Ning Yang and Mandy Xu.

Introduction

Sulfonation (also known as sulfurylation) is a significant phase II conjugation reaction that occurs throughout the metabolism of both xenobiotics and endogenous substances.

It is catalyzed by sulfotransferases (SULT). In humans, the eighteen SULT genes are divided into five gene families.

Recognizing the metabolic phenotyping and inhibition of sulfotransferases is essential for analyzing drug-drug interactions during drug discovery and development.

Conventional probe substrates, such as 4-nitrophenol and dopamine, frequently require ion-pairing reagents and can damage instruments, rendering them unsuitable for high-throughput LC-MS/MS analysis.

To address these constraints, this study utilized new probe substrates, developing a panel of in vitro phenotyping and inhibition tests to characterize SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1E1, and SULT2A1 via recombinant enzymes and optimized LC-MS/MS methodologies.

Conclusion

The researchers in this study designed and validated LC-MS/MS-based phenotyping assays for SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1E1, and SULT2A1. These assays allow for the assessment of compound substrate potential towards various SULT enzymes.

To improve the accuracy and throughput of SULT inhibition tests, probe substrates were selected and adjusted for each enzyme.

The relevant LCMS/MS procedures were also optimized. Key parameters such as probe substrate K_m and IC₅₀ values, as well as appropriate protein concentrations for recombinant SULT enzymes, were identified.

The validated in vitro SULT phenotyping and inhibition assays are now ready for use in evaluating compounds of interest during drug discovery and development.

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References

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Last Updated: Dec 18, 2025