Tandem mass spectrometric characterization of branched peptides derived from photoaffinity labeling

• Published in: International journal of mass spectrometry Vol. 228; no. 2; pp. 527 - 540
• Main Authors: Jahn, Olaf, Tezval, Hossein, Spiess, Joachim, Eckart, Klaus
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.08.2003
• Subjects: Benzophenone photoprobe; Collision-induced dissociation (CID); Corticotropin-releasing factor (CRF); Peptide–protein interaction; Benzophenone photoprobe; Collision-induced dissociation (CID); Corticotropin-releasing factor (CRF); Peptide–protein interaction
• ISSN: 1387-3806; 1873-2798
• DOI: 10.1016/S1387-3806(03)00159-3

Covalent photoadducts were obtained by photoaffinity labeling of the corticotropin-releasing factor (CRF) binding protein (CRFBP) with three photoreactive benzophenone-derivatized CRF analogs that differed in the position of the photophore. Enzymatic digestion of these photoadducts resulted in branched peptides that were analyzed by tandem mass spectrometry (MS/MS) under the conditions of high-energy collision-induced dissociation. Thereby, the branching site and, thus, the site of photoincorporation into CRFBP was identified. Comparison of the MS/MS spectra of the single linear peptide components with that of the branched peptide is presented as an analytical approach to facilitate its structural characterization. The major fragmentation pathways of the linear peptides were used to assist the assignment of the fragments derived from backbone fragmentation of the branched peptide. In addition, the low-mass fragment ions that are indicative for the presence of certain amino acid residues were analyzed to assign the modified amino acid residue. By using photoaffinity labeling in combination with mass spectrometry, three arginine residues in position 23, 36, and 111 of rat CRFBP were identified as the sites of photoincorporation of the three different photoreactive CRF analogs. Thereby, three contact sites between CRF and CRFBP could be assigned. These results in future will be helpful to further elucidate the conformation of the ligand-binding site of CRFBP.