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The non-covalent interactions between small drug molecules and disease-related proteins (ligand-target interactions) mediate various pharmacological processes in the treatment of different diseases. The development of the analytical methods to assess those interactions, including binding sites, binding energies, stoichiometry and association-dissociation constants, could assist in clarifying the mechanisms of action, precise treatment of targeted diseases as well as the targeted drug discovery. For the last decades, mass spectrometry (MS) has been recognized as a powerful tool to study the non-covalent interactions of the ligand-target complexes with the characteristics of high sensitivity, high-resolution, and high-throughput. Soft ionization mass spectrometry, especially the electrospray mass spectrometry (ESI-MS) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), could achieve the complete transformation of the target analytes into the gas phase, and subsequent detection of the small drug molecules and disease-related protein complexes, and has exerted great advantages for studying the drug ligands-protein targets interactions, even in case of identifying active components as drug ligands from crude extracts of medicinal plants. Despite of other analytical techniques for this purpose, such as the NMR and X-ray crystallography, this review highlights the principles, research hotspots and recent applications of the soft ionization mass spectrometry and its hyphenated techniques, including hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (CX-MS), and ion mobility spectrometry mass spectrometry (IMS-MS), in the study of the non-covalent interactions between small drug molecules and disease-related proteins.

The separation of high-purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two-dimensional reversed-phase preparative method was successfully developed for the separation of high-purity compounds from Oxytropis falcata. Based on the analysis results, an ODS C18 prep column was used for first-dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high-purity compounds in the second-dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans-cinnamic acid (1) and trifolirhizin (2) from Oxytropis falcata. This method has the potential to be an efficient separation method of high-purity compounds from Oxytropis falcata and it shows great promise for the separation of high-purity compounds from complex samples.