title: Characterization of peptide-protein interactions using photoaffinity labeling and LC/MS.
abstract: The combination of photoaffinity labeling (PAL) with modern mass spectrometric techniques is a powerful approach for the characterization of peptide-protein interactions. Depending on the analytical strategy applied, a PAL experiment can provide different levels of information ranging from the identification of interaction partners to the structural characterization of ligand-binding sites. On the basis of LC/MS data generated in the framework of the identification of the binding site of the neuropeptide corticotropin-releasing factor (CRF) on its binding protein (CRFBP), the key role of LC/MS in the characterization of photoadducts on different structural levels was demonstrated. Covalent photoadducts of rat CRFBP (rCRFBP) were obtained by PAL with different mono- and bifunctional benzophenone photoprobes designed on the basis of the sequence of the synthetic CRF fragment human/rat CRF(6-33) which binds to CRFBP with high affinity. In view of the stoichiometry, LC/MS analysis revealed that the photoadducts consisted of one molecule of photoprobe and one molecule of rCRFBP. For a further characterization of the photoadducts on the oligopeptide level, enzymatic digests of unlabeled rCRFBP and of the respective photoadduct were compared by peptide mapping monitored with LC/MS. Thereby, it was found that the photoprobe that contained the photophore at its N-terminus labeled the amino acid sequence rCRFBP(34-38), whereas the photoprobe that contained the photophore at its C-terminus labeled rCRFBP(12-26). On the basis of the characterization of the photoadduct formed by rCRFBP and the bifunctional photoprobe that contained photophores on both termini, semiquantitative comparison of different enzymatic digests was accomplished by application of the mass-selective multiple ion chromatogram strategy.
authors.affiliation: Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Goettingen, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Jahn
authors.forename: Klaus
authors.initials: K
authors.lastname: Eckart
authors.forename: Hossein
authors.initials: H
authors.lastname: Tezval
authors.forename: Joachim
authors.initials: J
authors.lastname: Spiess
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000737:chemistry; D002352:Carrier Proteins / Q000138:chemical synthesis / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D019920:Photoaffinity Labels / Q000737:chemistry*; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D051381:Rats
chemical_list: D001577:Benzophenones; D002352:Carrier Proteins; D010455:Peptides; D019920:Photoaffinity Labels; D011506:Proteins; C067133:corticotropin releasing factor-binding protein; C047723:benzophenone