Frontal affinity chromatography—mass spectrometry
- 2 August 2007
- journal article
- Published by Springer Nature in Nature Protocols
- Vol. 2 (8), 1907-1917
- https://doi.org/10.1038/nprot.2007.262
Abstract
Frontal affinity chromatography (FAC) is a biophysical method for the discovery and characterization of molecular interactions in a flow-based system. Several different modes of analysis are possible by interfacing to the mass spectrometer, including robust single-compound characterizations as well as high-throughput screening of over 1,000 compounds per run. The method supports thermodynamic and kinetic characterization of interactions for a wide range of molecular species and possesses similarities to flow-based biosensors such as surface plasmon resonance. It offers sensitive detection of ligands present well below their respective dissociation constants, and can be assembled from readily available laboratory components. Direct coupling of the FAC cartridge to the mass spectrometer is useful for the interrogation of single compounds or mixtures of limited complexity. An offline fractionation schema is more appropriate for discovery-mode applications. A high-performance FAC system enabling both modes can be assembled in 2-3 h. Measurements of dissociation constants can be made with such a system in 0.5-3 h, and the system supports higher-throughput screening modes at a rate of 10,000 compounds d(-1).Keywords
This publication has 30 references indexed in Scilit:
- Identification of P-glycoprotein substrates using open tubular chromatography on an immobilized P-glycoprotein column: Comparison of chromatographic results with Caco-2 permeabilityAnalytica Chimica Acta, 2006
- Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologiesCurrent Opinion in Biotechnology, 2006
- High-Throughput Screening for Enzyme Inhibitors Using Frontal Affinity Chromatography with Liquid Chromatography and Mass SpectrometryAnalytical Chemistry, 2005
- Capillary-Scale Frontal Affinity Chromatography/MALDI Tandem Mass Spectrometry Using Protein-Doped Monolithic Silica ColumnsAnalytical Chemistry, 2005
- Peer Reviewed: Biosensor Alternative: Frontal Affinity ChromatographyAnalytical Chemistry, 2004
- Protein-Doped Monolithic Silica Columns for Capillary Liquid Chromatography Prepared by the Sol−Gel Method: Applications to Frontal Affinity ChromatographyAnalytical Chemistry, 2004
- Displacement and Nonlinear Chromatographic Techniques in the Investigation of Interaction of Noncompetitive Inhibitors with an Immobilized α3β4 Nicotinic Acetylcholine Receptor Liquid Chromatographic Stationary PhaseAnalytical Chemistry, 2002
- Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface‐ and solution‐based biophysical methodsProtein Science, 2002
- Affinity Fusion Strategies for Detection, Purification, and Immobilization of Recombinant ProteinsProtein Expression and Purification, 1997
- Measurement of competitive adsorption isotherms by frontal chromatographyIndustrial & Engineering Chemistry Research, 1987