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.

High-Pressure Synthesized Lithium Pentazolate Compound Metastable under Ambient Conditions

“…5 The data were analyzed following procedures specifically designed for FIA with extremely small sample quantities injected along with unknown impurities. 32 In such cases, the ion current integrated over the sought-for m/z ratio with respect to time (before, during, and after sample injection) is plotted. Two LiN 5 samples recovered under ambient conditions were dissolved in methanol and injected in the mass spectrometer.…”

“…Methanol was chosen as the solvent since it had previously been shown to readily dissolve the pentazolate anion . The data were analyzed following procedures specifically designed for FIA with extremely small sample quantities injected along with unknown impurities . In such cases, the ion current integrated over the sought-for m / z ratio with respect to time (before, during, and after sample injection) is plotted.…”

Receptor–Ligand Interaction Measured by Inductively Coupled Plasma Mass Spectrometry and Selenium Labeling

“…In contrast with radiolabeling or fluorescence-labeling binding assays where the measurement can be directly performed from the plate, LC-ICP-MS analysis requires us to recover receptor-bound ligands for their further injection into the analytical device. This was achieved by the use of a dissociation buffer constituted of water supplemented with 1% of trifluoroacetic acid (TFA), which was previously assessed to perform efficient receptor denaturation as well as solubilization of whole dissociated ligands . Moreover, to ensure total analyte recovery, we scratched the inner of the wells with a spatula.…”

“…From a chemical point of view, selenium presents the advantage of being inserted into the analyte through conventional sulfur organic chemistry because both of these chalcogen atoms share similar physicochemical properties. Moreover, ICP-MS hyphenated to reversed-phase chromatography (RP-LC-ICP-MS) was very recently successfully investigated for the quantification purpose of selenopeptides. , Therefore, we chose such approach combining dedicated chemical labeling adapted to elemental MS analyses to achieve receptor–ligand interaction measurement. We developed the methodology with the well characterized V 1A subtype of vasopressin receptor (V 1A -R) implicated in a wide diversity of physiological processes such as vasoconstriction and emotional behavior and involving arginine vasopressin (AVP), a cyclic nonapeptide, as native ligand.…”

Chemical Analysis Program for the Time of Flight Mass Spectrometry System

“…Mass spectrometry is an analytical method and has a wide applications including; industrial quality control [1]- [3], pharmacology [4]- [7], isotope ratio determination [7]- [10] and space exploration researches [11][12][13]. Mass spectrometers consist of three units, which are an ionisation unit, separation unit and detector.…”