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Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells

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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.

A novel analytical workflow for the LC-MS-based metabolomic analysis of adherent cultured hepatic cells was developed. Three key steps were evaluated: a) cell harvesting, which includes cell detachment and metabolism quenching; b) metabolite extraction; and c) LC-MS analysis, assessing the use of RP and HILIC chromatographies. The final protocol allowed us to extend the metabolome coverage and enabled the detection of a wide range of metabolites from highly polar compounds to a wide range of lipids

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Acknowledgments

This work has been supported by the Instituto de Salud Carlos III of the Spanish Ministry of Science and Innovation (FIS PI14/00026 and FIS PI13/0986). A.L. is grateful for a Miguel Server II contract (CPII14/00004) from the above Ministry/Instituto de Salud Carlos III. J.C. G.-C. is grateful for a pre-doctoral contract from the Vali + d program of the Conselleria d’Educació (Regional Valencian Ministry of Education). S.L. is grateful for a contract (PTA2012-7224-I) from the Spanish Ministry of Economy and Competitiveness.

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Authors and Affiliations

  1. Unidad de Hepatología Experimental, Unidad Analítica, Instituto de Investigación Sanitaria, Fundación Hospital La Fe, Av. Fernando Abril Martorell 106, Valencia, 46026, Spain

    Juan Carlos García-Cañaveras, Silvia López, José Vicente Castell, M. Teresa Donato & Agustín Lahoz

  2. CIBERehd, Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, FIS, Barcelona, Spain

    Silvia López, José Vicente Castell & M. Teresa Donato

  3. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina y Odontología, Universidad de Valencia, Av. Blasco Ibáñez 15, 46010, Valencia, Spain

    Juan Carlos García-Cañaveras, José Vicente Castell & M. Teresa Donato

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  1. Juan Carlos García-Cañaveras
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  2. Silvia López
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  3. José Vicente Castell
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Correspondence to Agustín Lahoz.

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All the animals received human care and all experimental protocols were approved by the institutional animal ethics committee and performed in accordance with national and institutional regulations.

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García-Cañaveras, J.C., López, S., Castell, J.V. et al. Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells. Anal Bioanal Chem 408, 1217–1230 (2016). https://doi.org/10.1007/s00216-015-9227-8

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