Isotope and Affinity Tags in Photoreactive Substance P Analogues To Identify the Covalent Linkage within the NK-1 Receptor by MALDI-TOF Analysis

详细信息    查看全文

• 作者：Emmanuelle Sachon ; Olivier Tasseau ; Solange Lavielle ; Sandrine Sagan ; and Gé ; rard Bolbach
• 刊名：Analytical Chemistry
• 出版年：2003
• 出版时间：December 1, 2003
• 年：2003
• 卷：75
• 期：23
• 页码：6536 - 6543
• 全文大小：181K
• 年卷期：v.75,no.23(December 1, 2003)
• ISSN：1520-6882

文摘

Photoreactive analogues of substance P (biotin sulfone-spacer (amino pentanoic or Gly₃)-Arg-Pro-Lys-Pro-(pBzl)Phe-Gln-Phe-Phe-Gly-Leu-Met(O₂)NH₂) with or withoutisotope (deuterium) labeling have been synthesized. Deuteriums were present on (d)-biotin or epibiotin sulfone(D₃), on the Gly₃ spacer linker (D₆), or on the Gly inposition 9 of SP (D₂). Therefore, peptide analogues couldbe either unlabeled or tri-, penta-, or hexadeuterated.Results obtained with the use of these peptide analoguesshow that (d)-biotin sulfone and epibiotin sulfone are notrecognized 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 1:1molar ratio of nondeuterated and deuterated photoreactive substance P (SP) analogues in position 5, followedby combined digestions, purification, and MALDI-TOFmass spectrometry analysis, made the identification of thedomain of the receptor covalently linked by the photoreactive SP analogue easier. Indeed, doublets in massspectra were specific for the covalent complex whereassingle peaks could be attributed to contaminating species.This method is particularly suitable when minute amountsof complex have to be analyzed, as in the case of highlyhydrophobic G-protein coupled receptors.