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Frontal affinity chromatography—mass spectrometry

Nature Protocols volume 2, pages 1907–1917 (2007)Cite this article

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.

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Figure 1: The chromatogram in a frontal affinity chromatography (FAC) experiment.
Figure 2: Frontal affinity chromatography–mass spectrometry (FAC–MS) in discovery mode, demonstrating the detection of a ligand for human thrombin identified by online FAC–MS screening of a mixture of 100 tripeptides8.
Figure 3: Frontal affinity chromatography–mass spectrometry (FAC–MS) for ligand characterization; screening a racemic mixture of ligands arising from the synthesis of 4-hydroxy-2,2-dimethyl-cyclopent-1-yl 1-thio-β-D-galactopyranoside, m/z 331.1 (breakthrough curve in red)22.
Figure 4: Kd determination of GM1 pentasaccharide-cholera toxin-B interaction using the staircase method.
Figure 5: Schematic of the two operational modes of frontal affinity chromatography (FAC).
Figure 6: Hand-packing a frontal affinity chromatography (FAC) cartridge.
Figure 7: Valve configuration for the ligand characterization mode.

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Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, T2N 4N1, Alberta, Canada

    Ella S M Ng & David C Schriemer

  2. Defence R & D Canada—Suffield, P.O. Box 4000, Medicine Hat, T1A 8K6, Alberta, Canada

    Nora WC Chan

  3. Upchurch Scientific Inc., IDEX Health & Science, 619 Oak Street, Oak Harbor, Washington, USA

    Darren F Lewis

  4. Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen-Valby, DK-2500, Denmark

    Ole Hindsgaul

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  1. Ella S M Ng
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  3. Darren F Lewis
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  4. Ole Hindsgaul
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  5. David C Schriemer
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Corresponding author

Correspondence to David C Schriemer.

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Competing interests

As one of the original developers of the FAC systems is now employed at Upchurch Scientific, we have continued prototype development work with this company. They do not sell any FAC systems, but as a number of their individual components are used, we are declaring competing financial interests on behalf of this author (Dr. Darren Lewis). There are no competing financial interests for the other authors. Note that we have taken care to specific alternative suppliers of components where possible, to avoid a vendor bias.

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Ng, E., Chan, N., Lewis, D. et al. Frontal affinity chromatography—mass spectrometry. Nat Protoc 2, 1907–1917 (2007). https://doi.org/10.1038/nprot.2007.262

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