Pulsed Ultrafiltration Mass Spectrometry:  A New Method for Screening Combinatorial Libraries

Abstract

In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.

Analysis and screening of combinatorial libraries using mass spectrometry

“…A versatile approach to screening solution phase combinatorial libraries and natural product extracts is pulsed ultrafltration-mass spectrometry [57,58], which utilizes a standard LC-MS system with an ultrafiltration chamber substituted for the HPLC column. The principle of pulsed ultrafiltration screening of combinatorial libraries is shown in Figure 9.…”

“…After unbound compounds are removed, the hits from the library are eluted from the chamber by destabilizing the ligand-receptor complex using an organic solvent, a pH change, or a combination of both. The released ligands are identified on-line using APCI or electrospray mass spectrometry [57] or collected and analyzed off-line using mass spectrometry, LC-MS, or LC-MS-MS [59]. Figure 9.…”

Covalent modification of stathmin by CCNU determined by FTMS analysis of modified proteins and tryptic peptides

“…Mass spectrometry has been used as a tool for characterizing protein-small molecule interactions in solution [1][2][3][4][5][6]. One of the critical aspects of such studies is the ability to assess the extent and/or sites of small molecule incorporation.…”

Mass spectrometry and non-covalent protein-ligand complexes: Confirmation of binding sites and changes in tertiary structure

“…Ancillary techniques involving immobilized proteins [6], pulsed ultrafiltration [7], affinity chromatography [8,9] or size exclusion chromatography [10,11] as a means for isolating members of molecular complexes prior to MS analysis have also been developed to screen for potential protein-ligand complexes. Unlike the previous examples, these techniques do not rely on ionization and detection of intact complexes.…”