Investigation of labile protein-ligand interactions by native electrospray ionization mass spectrometry

Abstract

The investigations of noncovalent interaction between proteins (P) and small ligands (L) have played an important role for drug discovery and therapy. Electrospray ionization mass spectrometry (ESI-MS) has been typically applied to analysis of P-L interactions. However, some labile complexes are problematic for determination of association constants (Ka) by ESI-MS due to their in-source dissociation. Here, a strategy based on native MS for analysis of the labile complexes was developed. The reference ligand (Lref) with specific binding to P and a nonspecific binding ligand were employed. The equations for Ka calculation of labile complexes were developed. According to mathematical fitting curves, the Ka values of partially dissociated lysozyme-icariin complex and completely dissociated lysozyme-berberine complex were obtained. The obtained Ka values were consistent with those by fluorescence method. This strategy can be applied to the labile complexes of other small ligands to protein.