Isotope and Affinity Tags in Photoreactive Substance P Analogues To Identify the Covalent Linkage within the NK-1 Receptor by MALDI-TOF Analysis Emmanuelle Sachon, Olivier Tasseau, Solange Lavielle, Sandrine Sagan, and Ge ´rard Bolbach* UMR 7613 CNRSsUniversite ´ Pierre et Marie Curie, Structure et Fonction de Mole ´ cules Bioactives, case courrier 182, 4 Place Jussieu, 75252 Paris Cedex 05, France 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. Deu- teriums 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 photoreac- tive 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 photore- active 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. Among the different strategies used to elucidate the interaction domain of a ligand within a receptor protein, photolabeling 1,2 is particularly suitable and complementary to mutagenesis studies. After photolabeling, the ligand-receptor complex is subject to one or more enzymatic digestions, chemical cleavage, or both, subsequent purification step(s) being then required to recover the fragments of interest. Besides the design of the photoactivable probes, this purification step is often the cornerstone of such studies. Indeed, HPLC separation requires nanomole amounts of radioactive materials to overcome the adsorption losses. This quantity is a real hindrance to the study of hydrophobic proteins such as recombinant GTP binding-protein coupled receptors that are not easily purified in high amounts from mammal cells. A few years ago, we had developed a procedure involving biotin/ streptavidin purification, which does not require the use of a radioactive photoactivable ligand. 3,4 After enzymatic digestions, chemical cleavage, or both, the biotin present at the N-terminus of the photoactivable ligand is used to fish with streptavidin-coated magnetic beads the ligand-receptor complex out of the cellular magma. 4-6 Characterization of the digested complex is then achieved by mass spectrometry, mainly MALDI-TOF because of its high sensitivity (subpicomolar range), its high resolution, and great tolerance to biological media. Recent results using these successive steps have shown the interest and the capabilities of this procedure to define the interaction domain between SP and the human NK-1 (hNK-1) receptor. 4-6 Despite its apparent versatility and ability, this strategy must however be optimized. Indeed, because of the presence of peaks hard to interpret in the mass spectra, a lot of rather tedious blanks and control experiments are needed before getting final results. The problem is to determine whether these peaks are related to the photoreactive ligand-receptor complex or to contaminating peptides still present after the purification procedure. The fact that the enzyme used can sometimes cleave the receptor and the ligand, and that chymotrypsin-like activity always derives from trypsin during long-time incubations, 7 renders interpretation of the mass spectra even more complicated. Consequently, various enzymatic or chemical digestions and different digestion times are needed to interpret unambiguously the data obtained from mass spectrometry. We have now developed a strategy complementary to the procedure we used so far. This method, derived from the isotope- coded affinity tag method in proteomic studies, 8,9 is based on the isotopic (deuterium) labeling of the photoactivable ligand, to get * To whom correspondence should be addressed. E-mail: bolbach@ ccr.jussieu.fr. Telephone: (33) 1 44 27 31 80. Fax: (33) 1 44 27 38 43. (1) Dorman, G.; Prestwich, G. D. Trends Biotechnol. 2000 , 18, 64-77. (2) Hatanaka, Y.; Sadakane, Y. Curr. Top. Med. Chem. 2002 , 2, 271-288. (3) Girault, S.; Chassaing, G.; Blais, J.-C.; Brunot, A.; Bolbach, G. Anal. Chem. 1996 , 68, 2122-2126. (4) Girault, S.; Sagan, S.; Bolbach, G.; Lavielle, S.; Chassaing, G. Eur. J. Biochem. 1996 , 240, 215-222. (5) Lequin, O.; Bolbach, G.; Frank, F.; Convert, O.; Girault-Lagrange, S.; Chassaing, G.; Lavielle, S.; Sagan, S. J. Biol. Chem. 2002 , 277, 22386-22394. (6) Sachon, E.; Bolbach, G.; Chassaing, G.; Lavielle, S.; Sagan, S. J. Biol. Chem. 2002 , 277, 50409-50414. (7) Keil-Dlouha, V.; Zylber, N.; Tong, N.; Keil, B. 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