Identification of Active Herbal Ingredients by Affinity Selection Mass Spectrometry Coupled with Ultrafiltration

摘要

Natural product, derived from various resources, is now still the mainstay in the world for preventing and treating disease. Finding new leads to macromolecular targets among complex extracts of natural products requires a selective screening assay to reduce time, cost, and laborious. Affinity selection mass spectrometry (ASMS), which does not depend on drug target function for their readout, outperform the traditional bioassay-guided isolation method in screening complex mixture of potential ligands. As most types of target proteins are soluble, ultrafiltration combined with liquid chromatography-mass spectrometry (LC-MS) technique is one of the ASMS techniques that is particular useful because the protein is maintained in solution during binding and screening. The general protocol of the ultrafiltration-based ASMS approach is shown in Fig. 1. In this technique, a certain amount of centrifugal force is applied to the ultrafiltration chamber-equipped centrifugal tube, which contains a molecular weight cutoff (MWCO) membrane for excluding solvents as well as unbound small molecules but retaining proteins and protein-ligand complexes after the binding of the ligands to the target protein is equilibrated. After filtration, the retentate would protein and protein-ligand complexes only, as an appropriate filter device with the right MWCO is selected. The retentate was successively restored to the initial volume. After another two rounds of filtration, the concentration of unbound compound in the protein-containing chamber will be negligible in comparison to the concentration of bound ligands. Hence, bound ligands are easily identified by ESI mode MS after the dissociation of the protein-ligand complexes. According to the detection principle, we have analyzed and identified potential ligands using LC-MS after the ligand-protein complexes are dissociated. Experimentally, a candidate with a relative binding value (Eq.1) higher than 20% would be considered as a possible bound ligand requiring a retest of screening. And the false positive results due to the aggregation or non-specific binding can be determined according to the retention factor (Eq.2).