Hyphenation of Affinity Capillary Electrophoresis with Mass Spectrometry for the Study of Ligand–Protein Interactions: n‑Methylmorpholine Acetate Buffer and Polydopamine-Based Coating as Key Assets

The direct and precise assessment of ligand–protein interactions under nearly physiological conditions is the core of drug discovery. In this context, affinity capillary electrophoresis (ACE) has become an emerging and reliable approach. The hyphenation of ACE with mass spectrometry (MS) is even more powerful than the classical ACE-UV methodology. It reduces compound identification errors and increases throughput by facilitating the analysis of the mixtures. However, buffers and capillary coatings compatible with mass spectrometry and operating under physiological conditions are very limited. In this paper, n-methylmorpholine acetate buffer and polydopamine-based coating were highlighted as major assets for CE-MS studies involving native proteins. Thanks to its protein desorption property, n-methylmorpholine improved the peak shape of proteins during CE analysis at physiological pH. The polydopamine-based neutral coating developed in this study is simple to prepare and demonstrated high stability at pH 7.4, enabling its use with an MS detector. The combination of these two key elements enabled us to successfully convert our ACE-UV method for coagulation factor XIIa into an ACE-MS approach operating at physiological pH. This study extends the scope of ACE for medicinal chemistry projects.