Hyphenation of affinity capillary electrophoresis in near- physiological conditions with mass spectrometry for drug discovery

The drug discovery process requires the precise determination of the affinity between small molecules and the protein target under physiological conditions. In this context, an emerging and reliable approach is affinity capillary electrophoresis (ACE). To expand its prospects, it is necessary to couple it to a mass spectrometer (MS). This detector can reduce compound identification errors and increase the analysis throughput by allowing the analysis of mixtures. However, there are very few buffers and capillary coatings that are compatible with MS under physiological conditions. To enable the coupling of ACE with MS, we first investigated the properties of the volatile buffer n-methylmorpholine acetate by CE-UV. We studied the electrophoretic behavior of small molecules and
proteins on bare fused silica capillaries. We observed that n-methylmorpholine enhances the peak shape of proteins. Then, by exploiting the characteristics of polydopamine, we optimized a neutral coating based on poly(N-isopropylacrylamide), easy to produce and permanent at pH 7.4. Finally, our best conditions were validated by determining the dissociation constant of p-aminobenzamidine with coagulation factor XIIa by ACE-MS.
Protein adsorption to silica is a common problem in CE, leading to deterioration of repeatability, peak shape, and resolution. Typically, harsh treatments under extremely acidic conditions are used. Desorption by n-methylmorpholine occurs under mild conditions. Moreover, n-methylmorpholine is volatile. Co-deposition of poly(N-isopropylacrylamide) and polydopamine generates stable coatings at physiological pH and can therefore be used with MS. The combination of these two key elements
allowed us to couple our ACE method with MS. This study increases the applicability of ACE in the context of the drug discovery process [1].
[1] C. Davoine, M. Fillet, 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, Anal. Chem. 97 (2025) 3988–3995. https://doi.org/10.1021/acs.analchem.4c05559.