Ligand-protein interaction’s monitoring by CE-MS in the context of drug development: BGE and capillary coating optimizations

Previously, our team developed affinity capillary electrophoresis (ACE) methods using a DAD as a detector [1–3]. ACE proved to be a reliable approach for validating fragment hits and determining their dissociation constants [1,2]. The next step in this project was to develop an ACE method compatible with MS. Indeed, MS detection can extend the applicability of our approach to compounds with little or no UV-vis absorption. MS also enables the confirmation of the ligand identity. This additional output avoids misinterpretation of the results (false structural assignment of binders), which is a valuable asset during a drug discovery campaign. However, detection by MS requires a volatile buffer and a permanent coating. Our previous methods used a Tris-Hepes buffer and a dynamic PEO coating, which were not compatible with MS detectors. Overall, to study ligand-protein interactions under near-physiological conditions using CE-MS, we need a volatile BGE buffering at pH 7.4 and a permanent, antifouling coating stable at physiological pH. The antifouling coating is mandatory when studying ligand-protein interactions, as interactions of the protein and/or ligand with the capillary wall decrease their free concentration in solution, leading to a bias when assessing affinity. In this work, we studied protein adsorption on silica using soft and hard proteins of different molecular weights, as well as small molecules that mimic ligands with various acid/base and hydrophilic characteristics. We evaluated the properties of n-methylmorpholine (NMM) as a buffer for CE-MS applications requiring physiological conditions. We observed that NMM in itself can reduce protein adsorption to silica. We also synthesized and investigated poly(n-isopropylacrylamide)-grafted polydopamine (PDA) coatings for CE-MS interaction studies. By monitoring the peak shapes and migration times’ stability, we studied several critical parts of the protocol, such as the PDA deposition (grafting vs one-pot deposition), the end capping, and the storage conditions.

[1] E. Farcaş, C. Bouckaert, A.C. Servais, J. Hanson, L. Pochet, M. Fillet, Partial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin, Anal. Chim. Acta. 984 (2017) 211–222. https://doi.org/10.1016/j.aca.2017.06.035.
[2] C. Davoine, M. Fillet, L. Pochet, Capillary electrophoresis as a fragment screening tool to cross-validate hits from chromogenic assay: Application to FXIIa, Talanta. 226 (2021) 122163. https://doi.org/10.1016/j.talanta.2021.122163.
[3] C. Davoine, A. Pardo, L. Pochet, M. Fillet, Fragment Hit Discovery and Binding Site Characterization by Indirect Affinity Capillary Electrophoresis: Application to Factor XIIa, Anal. Chem. 93 (2021) 14802–14809. https://doi.org/10.1021/acs.analchem.1c03611.