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 (K_a) 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 (L_ref) with specific binding to P and a nonspecific binding ligand were employed. The equations for K_a calculation of labile complexes were developed. According to mathematical fitting curves, the K_a values of partially dissociated lysozyme-icariin complex and completely dissociated lysozyme-berberine complex were obtained. The obtained K_a values were consistent with those by fluorescence method. This strategy can be applied to the labile complexes of other small ligands to protein.

蛋白质（P）和小配体（L）之间非共价相互作用的研究对于药物发现和治疗起了重要作用。电喷雾电离质谱（ESI-MS）通常已用于分析PL相互作用。然而，一些不稳定的络合物是用于测定缔合常数（K的问题_一）通过ESI-MS由于它们在源离解。在这里，开发了一种基于天然MS的分析不稳定复合物的策略。使用对P具有特异性结合的参考配体（L _ref）和非特异性结合配体。开发了用于计算不稳定配合物的K _a的方程。根据数学拟合曲线，K _a获得了部分解离的溶菌酶-大豆素复合物和完全解离的溶菌酶-小ber碱复合物的值。所得到的K _a值与荧光法一致。该策略可应用于其他小的配体与蛋白质的不稳定复合物。