title: Investigation of Elemental Mass Spectrometry in Pharmacology for Peptide Quantitation at Femtomolar Levels.
abstract: In the search of new robust and environmental-friendly analytical methods able to answer quantitative issues in pharmacology, we explore liquid chromatography (LC) associated with elemental mass spectrometry (ICP-MS) to monitor peptides in such complex biological matrices. The novelty is to use mass spectrometry to replace radiolabelling and radioactivity measurements, which represent up-to now the gold standard to measure organic compound concentrations in life science. As a proof of concept, we choose the vasopressin (AVP)/V1A receptor system for model pharmacological assays. The capacity of ICP-MS to provide highly sensitive quantitation of metallic and hetero elements, whatever the sample medium, prompted us to investigate this technique in combination with appropriate labelling of the peptide of interest. Selenium, that is scarcely present in biological media, was selected as a good compromise between ICP-MS response, covalent tagging ability using conventional sulfur chemistry and peptide detection specificity. Applying selenium monitoring by elemental mass spectrometry in pharmacology is challenging due to the very high salt content and organic material complexity of the samples that produces polyatomic aggregates and thus potentially mass interferences with selenium detection. Hyphenation with a chromatographic separation was found compulsory. Noteworthy, we aimed to develop a straightforward quantitative protocol that can be performed in any laboratory equipped with a standard macrobore LC-ICP-MS system, in order to avoid time-consuming sample treatment or special implementation of instrumental set-up, while allowing efficient suppression of all mass interferences to reach the targeted sensitivity. Significantly, a quantification limit of 57 ng Se L-1 (72 femtomoles of injected Se) was achieved, the samples issued from the pharmacological assays being directly introduced into the LC-ICP-MS system. The established method was successfully validated and applied to the measurement of the vasopressin ligand affinity for its V1A receptor through the determination of the dissociation constant (Kd) which was compared to the one recorded with conventional radioactivity assays.
authors.affiliation: Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier, CNRS, ENSCM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Cordeau
authors.affiliation: Laboratoire de Chimie Analytique Bio-inorganique et Environnement LCABIE-IPREM, UMR 5254, Hélioparc, 2 av. Pr. Angot, 64053 Pau, France.
authors.forename: Carine
authors.initials: C
authors.lastname: Arnaudguilhem
authors.affiliation: Laboratoire de Chimie Analytique Bio-inorganique et Environnement LCABIE-IPREM, UMR 5254, Hélioparc, 2 av. Pr. Angot, 64053 Pau, France.
authors.forename: Brice
authors.initials: B
authors.lastname: Bouyssiere
authors.affiliation: Institute of Analytical Sciences (ISA), UMR 5280, CNRS, Université Lyon 1, 5 rue de la Doua, 69100 Villeurbanne, France.
authors.forename: Agnès
authors.initials: A
authors.lastname: Hagège
authors.affiliation: Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier, CNRS, ENSCM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.
authors.forename: Jean
authors.initials: J
authors.lastname: Martinez
authors.affiliation: Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier, CNRS, ENSCM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Subra
authors.affiliation: Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier, CNRS, ENSCM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.
authors.forename: Sonia
authors.initials: S
authors.lastname: Cantel
authors.affiliation: Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier, CNRS, ENSCM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.
authors.forename: Christine
authors.identifier: http://orcid.org/0000-0003-4646-4583
authors.initials: C
authors.lastname: Enjalbal
mesh_terms: D002853:Chromatography, Liquid; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010600:Pharmacology / Q000379:methods* / Q000592:standards*; D011485:Protein Binding; D012643:Selenium / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D014667:Vasopressins / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D014667:Vasopressins; D012643:Selenium