Evaluation of a micro volume pulsed ultrafiltration cell for screening ligands in non-covalent complexes.

A pulsed ultrafiltration cell with a 35 microL binding chamber was evaluated for its ability to screen ligands that formed non-covalent complexes with protein targets. The cell was tested with ligands to the targets of carbonic anhydrase and serum albumin. Non-covalent ligand binding to both of these targets was observed and bound ligands were eluted from the cell in less than five min. The cell was also demonstrated to effectively screen a methanolic fermentation broth extract spiked with a known inhibitor to carbonic anhydrase. In addition to detecting specific binding events, the pulsed ultrafiltration method was investigated for its ability to distinguish non-specific binding events. Using carbonic anhydrase with the zinc-binding site removed, it was found that non-specific complexes observed when using electrospray ionization alone were not detected when using the pulsed ultrafiltration mass spectrometry method.