| Affinity Selective Mass Spectrometry(ASMS)is a high throughput screening method using mass spectrometry to identify the compounds binding to the target protein.It has been widely used in ligand screening of various proteins.The Tip-based ASMS method was developed by our lab previously,and we optimized the buffer composition to reduce the non-specific adsorption of compounds to the beads,and optimized the workflow for processing mass spectrometry data in this study.The nonionic detergent was adding to the wash buffer in order to reduce the nonspecific adsorption.The low concentration of nonionic detergent in the wash buffer could not affect the activity of target protein,but it could wash away the nonspecifically-absorbed compound,reducing the false negative results and increasing the success rate of ASMS screening.We also introduce the data process workflow of targeted metabolomics to optimized the mass spectrometry data processing workflow of the Tip-based ASMS.We built a compound database with compound name,chemical form,calculated molecular weight etc.for identifying the compound from the mass spectrometry data.Compared with the previous workflow,the current one can increase the identification rate from 60% to 90%.In this study,the Tip-based ASMS method was applied to the compound screening of HSP90-N protein.We screened 2395 compounds,including FDA-approved clinical and preclinical compounds,and found the positive compound Dipyridamole.The antitumor mechanism of Dipyridamole was preliminarily explored by DIA proteomics. |
