Affinity capillary electrophoresis in near-physiological conditions coupled to mass spectrometry for ligand-protein interaction’s assessment in the context of drug discovery

Assessing interactions between small molecules and proteins is critical to discover new medicines. In this context, affinity capillary electrophoresis (ACE) emerges as a reliable approach for validating fragment hits and determining their dissociation constants [1–3]. The advantages of ACE include low sample and target consumption, its separative character, and the absence of target modification [4]. The ACE methods previously developed by our team [1–3] employed a DAD as a detector, which narrows its applicability to compounds with UV-Vis absorption. Therefore, we aimed to develop ACE methods compatible with mass spectrometry (MS) to extend its scope. MS increases throughput by
facilitating the analysis of mixtures and reduces compound identification errors, which are valuable assets during a drug discovery campaign. However, capillary coatings and buffers compatible with MS and operating under physiological conditions are highly limited. The Tris-Hepes buffer and dynamic PEO coating that we previously used [1–3] are incompatible with the MS detector. This work highlighted the value of n-methylmorpholine acetate buffer and polydopamine-based coating for CE-
MS studies involving native proteins. In the n-methylmorpholine acetate buffer, the peak shape of proteins during electromigration was improved. This amelioration was attributed to the ability of n-methylmorpholine to desorb proteins from the capillary surface. Regarding the development of MS-compatible coatings, we synthesized and studied poly(n-isopropylacrylamide)-grafted polydopamine coatings by investigating several critical parts of the protocol, such as the deposition (grafting vs one-pot deposition), the end-capping, and the storage conditions. The optimized polydopamine-based neutral coating is simple to prepare and demonstrated high stability at pH 7.4, enabling its use with an MS detector. Thanks to these two major assets, we successfully convert our ACE-UV method for coagulation factor XIIa into an ACE-MS approach operating at physiological pH. This work broadens the scope of this technology for drug discovery projects.
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Characterization by Indirect Affinity Capillary Electrophoresis: Application to Factor XIIa, Anal. Chem. 93 (2021) 14802–14809. https://doi.org/10.1021/acs.analchem.1c03611.
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characterizing noncovalent interactions for fragment-based drug discovery, TrAC Trends Anal. Chem. 166 (2023) 117161. https://doi.org/10.1016/j.trac.2023.117161.