Direct screening of natural product extracts using mass spectrometry.

The authors describe first a proof-of-concept experiment to show direct affinity screening using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) is a rapid and informative approach for natural product extract screening. The study used 10 alkaloid-enriched plant extracts and 8 desalted marine extracts spiked with specific inhibitors of bovine carbonic anhydrase II (bCAII; EC4.2.1.1) as a model set. The spiked extracts were incubated with bCAII and then analyzed by ESI-FTICR-MS. The noncovalent complexes were detected, and the specific inhibitors were reidentified in the spiked natural product extracts. There was no interference from the desalted/alkaloid-enriched extracts to the formation of the noncovalent complexes. The method allowed quick identification of the molecular mass of the bound ligand. The authors then applied the screening to identify active compounds in natural product extracts. They employed direct infusion and online size exclusion chromatography (SEC) ESI-FTICR-MS to detect intact target-ligand complex. Eighty-five methanolic plant extracts were screened against bCAII by direct infusion ESI-FTICR-MS and by online SEC-ESI-FTICR-MS. One noncovalent complex was identified from the same plant extract by both methods. The molecular weight of the bound ligand from this extract was determined. Mass-directed purification gave 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one (1) as the active compound. Subsequently, the binding to bCAII was confirmed by ESI-FTICR-MS. The binding specificity was determined by competition experiments between 1 and furosemide, a specific ligand of bCAII.