title: Isotope and affinity tags in photoreactive substance P analogues to identify the covalent linkage within the NK-1 receptor by MALDI-TOF analysis.
abstract: Photoreactive analogues of substance P (biotin sulfone-spacer (amino pentanoic or Gly(3))-Arg-Pro-Lys-Pro-(pBzl)Phe-Gln-Phe-Phe-Gly-Leu-Met(O(2))NH(2)) with or without isotope (deuterium) labeling have been synthesized. Deuteriums were present on (d)-biotin or epibiotin sulfone (D(3)), on the Gly(3) spacer linker (D(6)), or on the Gly in position 9 of SP (D(2)). Therefore, peptide analogues could be either unlabeled or tri-, penta-, or hexadeuterated. Results obtained with the use of these peptide analogues show that (d)-biotin sulfone and epibiotin sulfone are not recognized with the same affinity by streptavidin, with (d)-biotin sulfone displaying better affinity for the protein. Photolabeling of the human NK-1 receptor with a 1:1 molar ratio of nondeuterated and deuterated photoreactive substance P (SP) analogues in position 5, followed by combined digestions, purification, and MALDI-TOF mass spectrometry analysis, made the identification of the domain of the receptor covalently linked by the photoreactive SP analogue easier. Indeed, doublets in mass spectra were specific for the covalent complex whereas single peaks could be attributed to contaminating species. This method is particularly suitable when minute amounts of complex have to be analyzed, as in the case of highly hydrophobic G-protein coupled receptors.
authors.affiliation: UMR 7613 CNRS-Université Pierre et Marie Curie, Structure et Fonction de Molécules Bioactives, case courrier 182, 4 Place Jussieu, 75252 Paris Cedex 05, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Sachon
authors.forename: Olivier
authors.initials: O
authors.lastname: Tasseau
authors.forename: Solange
authors.initials: S
authors.lastname: Lavielle
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
authors.forename: Gérard
authors.initials: G
authors.lastname: Bolbach
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D018040:Receptors, Neurokinin-1 / Q000032:analysis* / Q000096:biosynthesis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013373:Substance P / Q000031:analogs & derivatives* / Q000032:analysis* / Q000096:biosynthesis
chemical_list: D018040:Receptors, Neurokinin-1; D013373:Substance P