title: Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.
abstract: MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.
authors.affiliation: Unidad de Hepatología Experimental, Unidad Analítica, Instituto de Investigación Sanitaria, Fundación Hospital La Fe, Av. Fernando Abril Martorell 106, Valencia, 46026, Spain.|Departamento de Bioquímica y Biología Molecular, Facultad de Medicina y Odontología, Universidad de Valencia, Av. Blasco Ibáñez 15, 46010, Valencia, Spain.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: García-Cañaveras
authors.affiliation: Unidad de Hepatología Experimental, Unidad Analítica, Instituto de Investigación Sanitaria, Fundación Hospital La Fe, Av. Fernando Abril Martorell 106, Valencia, 46026, Spain.|CIBERehd, Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, FIS, Barcelona, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: López
authors.affiliation: Unidad de Hepatología Experimental, Unidad Analítica, Instituto de Investigación Sanitaria, Fundación Hospital La Fe, Av. Fernando Abril Martorell 106, Valencia, 46026, Spain.|CIBERehd, Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, FIS, Barcelona, Spain.|Departamento de Bioquímica y Biología Molecular, Facultad de Medicina y Odontología, Universidad de Valencia, Av. Blasco Ibáñez 15, 46010, Valencia, Spain.
authors.forename: José Vicente
authors.initials: JV
authors.lastname: Castell
authors.affiliation: Unidad de Hepatología Experimental, Unidad Analítica, Instituto de Investigación Sanitaria, Fundación Hospital La Fe, Av. Fernando Abril Martorell 106, Valencia, 46026, Spain.|CIBERehd, Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, FIS, Barcelona, Spain.|Departamento de Bioquímica y Biología Molecular, Facultad de Medicina y Odontología, Universidad de Valencia, Av. Blasco Ibáñez 15, 46010, Valencia, Spain.
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Donato
authors.affiliation: Unidad de Hepatología Experimental, Unidad Analítica, Instituto de Investigación Sanitaria, Fundación Hospital La Fe, Av. Fernando Abril Martorell 106, Valencia, 46026, Spain. agustin.lahoz@uv.es.
authors.forename: Agustín
authors.initials: A
authors.lastname: Lahoz
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D003584:Cytological Techniques; D056945:Hep G2 Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D059625:Liquid-Liquid Extraction / Q000379:methods; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D057188:Workflow
keywords: Bioanalytical methods; HPLC; LC-MS; Mass spectrometry; Metabolomics; Process analysis; Sampling