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Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors

Nature Protocols volume 2, pages 119–130 (2007)Cite this article

Abstract

Among the main objectives of biomedical and proteomic research is to identify non-covalent interactions involving proteins. Here we provide a detailed protocol to apply matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry for such a purpose using proteases and protease inhibitors in complex biological samples. Our methodology is based on monitoring the reduction in intensity of inhibitors' mass spectrometric signals when their protease target is added to the MALDI sample. The versatility of the protocol permits the target to be added in a soluble form (direct protocol) or immobilized form (indirect protocol). The 'intensity fading' phenomenon is greatly favored when the binding assay is carried out in the sub-micromolar range and the interacting partners occur in mixtures of non-binding compounds. This protocol can be completed in 10 h, taking 20 or 30 min per sample to perform the mass spectrometric data acquisition, depending on whether a soluble or an immobilized target is used.

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Figure 1: Flowchart of the direct and indirect protocols.
Figure 2: Intensity fading mass spectrometry (MS) in model mixtures.
Figure 3
Figure 4: Intensity fading mass spectrometry (MS)—indirect protocol steps.
Figure 5: Intensity fading MS—direct protocol.
Figure 6: Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectra of three different RP-HPLC fractions from the leech extract showing inhibitory activity against trypsin, before (upper) and after (lower) the addition of the anhydrotrypsin beads.
Figure 7: Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectra of four different RP-HPLC fractions from the leech extract showing inhibitory activity against trypsin, before (upper) and after (middle) the addition of anhydrotrypsin beads.
Figure 8: Intensity fading mass spectrometry (MS) vs.

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Acknowledgements

We are very grateful to Professors Luís Serrano (EMBL, Germany), Peter Roepstorff and Thomas J.D. Jørgensen (University of Southern Denmark), Renato Zenobi (ETH Zürich, Switzerland) and M. Angeles Chavez (University of La Habana, Cuba), and their research teams, for their invaluable advice, collaboration and interest in the research and development of different aspects of the intensity fading MS methodology. Also to PROTEORED (Spain). This work has been supported by grants GEN2003-20642-C09-05 and BIO2004-05879 (Ministerio de Ciencia y Tecnología, Spain), grant LSHG-2006-018830-CAMP (EC-Dir.F), and BIO2004-05879, and by the Centre de Referència en Biotecnologia (Generalitat de Catalunya, Spain). O.Y. acknowledges a fellowship from the Ministerio de Ciencia y Tecnología.

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Authors and Affiliations

  1. Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica, Universitat Autònoma de Barcelona, Bellaterra (Barcelona), 08193, Spain

    Oscar Yanes, Enrique Querol & Francesc X Aviles

  2. Protein Center and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, 10021, New York, USA

    Josep Villanueva

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  1. Oscar Yanes
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  4. Francesc X Aviles
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O.Y. and J.V. contributed equally to this work.

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Correspondence to Francesc X Aviles.

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Yanes, O., Villanueva, J., Querol, E. et al. Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors. Nat Protoc 2, 119–130 (2007). https://doi.org/10.1038/nprot.2006.487

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