title: A Nontargeted UHPLC-HRMS Metabolomics Pipeline for Metabolite Identification: Application to Cardiac Remote Ischemic Preconditioning.
abstract: In recent years, the number of investigations based on nontargeted metabolomics has increased, although often without a thorough assessment of analytical strategies applied to acquire data. Following published guidelines for metabolomics experiments, we report a validated nontargeted metabolomics strategy with pipeline for unequivocal identification of metabolites using the MSMLS molecule library. We achieved an in-house database containing accurate m/z values, retention times, isotopic patterns, full MS, and MS/MS spectra. A UHPLC-HRMS Q-Exactive method was developed, and experimental variations were determined within and between 3 experimental days. The extraction efficiency as well as the accuracy, precision, repeatability, and linearity of the method were assessed, the method demonstrating good performances. The methodology was further blindly applied to plasma from remote ischemic pre-conditioning (RIPC) rats. Samples, previously analyzed by targeted metabolomics using completely different protocol, analytical strategy, and platform, were submitted to our analytical pipeline. A combination of multivariate and univariate statistical analyses was employed. Selection of putative biomarkers from OPLS-DA model and S-plot was combined to jack-knife confidence intervals, metabolites' VIP values, and univariate statistics. Only variables with strong model contribution and highly statistical reliability were selected as discriminated metabolites. Three biomarkers identified by the previous targeted metabolomics study were found in the current work, in addition to three novel metabolites, emphasizing the efficiency of the current methodology and its ability to identify new biomarkers of clinical interest, in a single sequence. The biomarkers were identified to level 1 according to the metabolomics standard initiative and confirmed by both RPLC and HILIC-HRMS.
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.
authors.forename: Judith
authors.identifier: 0000-0003-4987-1889
authors.initials: J
authors.lastname: Kouassi Nzoughet
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Bocca
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.|Département de Biochimie et Génétique, Centre Hospitalier Universitaire , 49933 Angers CEDEX 9, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Simard
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.|Département de Biochimie et Génétique, Centre Hospitalier Universitaire , 49933 Angers CEDEX 9, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Prunier-Mirebeau
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.|Département de Biochimie et Génétique, Centre Hospitalier Universitaire , 49933 Angers CEDEX 9, France.
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: Chao de la Barca
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.|Département de Biochimie et Génétique, Centre Hospitalier Universitaire , 49933 Angers CEDEX 9, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Bonneau
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.|Département de Biochimie et Génétique, Centre Hospitalier Universitaire , 49933 Angers CEDEX 9, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Procaccio
authors.affiliation: Institut MITOVASC, Laboratoire EA3860, Cardioprotection, Remodelage et Thrombose , Rue Haute de Reculée, FR-49045, Angers, France.|Département de Cardiologie, Centre Hospitalier Universitaire , 49933 Angers CEDEX 9, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Prunier
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.
authors.forename: Guy
authors.initials: G
authors.lastname: Lenaers
authors.affiliation: PREMMi, Pôle de Recherche et d'Enseignement en Médecine Mitochondriale, Institut MITOVASC, CNRS 6214, INSERM U1083, Université d'Angers , 4 Rue Larrey, 49933 Angers CEDEX 9, France.|Département de Biochimie et Génétique, Centre Hospitalier Universitaire , 49933 Angers CEDEX 9, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Reynier
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016208:Databases, Factual; D006801:Humans; D019157:Ischemic Preconditioning, Myocardial; D057230:Limit of Detection; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D055432:Metabolomics; D017208:Rats, Wistar; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers