Enantioselective Protein Affinity Selection Mass Spectrometry (EAS-MS)

Wang, X.; Sun, J.; Ahmad, S.; Yang, D.; Li, F.; Chan, U. H.; Zeng, H.; Simoben, C. V.; Houliston, S.; Dong, A.; Bolotokova, A.; Gibson, E.; Kutera, M.; Ghiabi, P.; Kondratov, I.; Matviyuk, T.; Chuprina, A.; Mavridi, D.; Lenz, C.; Joerger, A. C.; Brown, B. D.; Heath, R. B.; Yue, W.; Robbie, L. K.; Beyett, T. S.; Muller, S.; Knapp, S.; Harding, R. J.; Schapira, M.; Brown, P. J.; Santhakumar, V.; Ackloo, S.; Arrowsmith, C. H.; Edwards, A.; Peng, H.; Halabelian, L.

doi:10.1101/2025.01.17.633682

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bioRxiv · 10.1101/2025.01.17.633682

Enantioselective Protein Affinity Selection Mass Spectrometry (EAS-MS)

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Abstract

We report an enantioselective protein affinity selection mass spectrometry screening approach (E-ASMS) that enables the detection of weak binders, informs on selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screened 31 human proteins against a designed library of 8,210 chiral compounds. 16 binders to 12 targets, including many proteins predicted to be "challenging to ligand", were discovered and confirmed in orthogonal biophysical assays. 7 binders to 6 targets bound in an enantioselective manner, with KD values ranging from 3 to 20 {micro}M. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) were selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanism for enantioselectivity was readily explained. We conclude E-ASMS can be used to identify and characterize selective and weakly-binding ligands for novel protein targets with unprecedented throughput and sensitivity.

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Wang, X., Sun, J., Ahmad, S., Yang, D., Li, F., Chan, U. H., Zeng, H., Simoben, C. V., Houliston, S., Dong, A., Bolotokova, A., Gibson, E., Kutera, M., Ghiabi, P., Kondratov, I., Matviyuk, T., Chuprina, A., Mavridi, D., Lenz, C., Joerger, A. C., Brown, B. D., Heath, R. B., Yue, W., Robbie, L. K., Beyett, T. S., Muller, S., Knapp, S., Harding, R. J., Schapira, M., Brown, P. J., Santhakumar, V., Ackloo, S., Arrowsmith, C. H., Edwards, A., Peng, H., Halabelian, L.. 2025-01-22. Enantioselective Protein Affinity Selection Mass Spectrometry (EAS-MS). https://doi.org/10.1101/2025.01.17.633682

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