title: Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors.
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.
authors.affiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
authors.forename: Oscar
authors.initials: O
authors.lastname: Yanes
authors.forename: Josep
authors.initials: J
authors.lastname: Villanueva
authors.forename: Enrique
authors.initials: E
authors.lastname: Querol
authors.forename: Francesc X
authors.initials: FX
authors.lastname: Aviles
mesh_terms: D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D011480:Protease Inhibitors; D010447:Peptide Hydrolases