title: Metabolomic profiling of cultured cancer cells.
abstract: Quantitative proteomics approaches have been developed-and now begin to be implemented on a high-throughput basis-to fill-in the large gap between the genomic/transcriptomic setup of (cancer) cells and their phenotypic/behavioral traits, reflecting a significant degree of posttranscriptional regulation in gene expression as well as a robust posttranslational regulation of protein function. However, proteomic profiling assays not only fail to detect labile posttranslational modifications as well as unstable protein-to-protein interactions but also are intrinsically incapable of assessing the enzymatic activity, as opposed to the mere abundance, of a given protein. Thus, determining the abundance of theoretically all the metabolites contained in a cell/tissue/organ/organism may significantly improve the informational value of proteomic approaches. Several techniques have been developed to this aim, including high-performance liquid chromatography (HPLC) coupled to quadrupole time-of-flight (Q-TOF) high-resolution mass spectrometry (HRMS). This approach is particularly advantageous for metabolomic profiling as it offers elevated accuracy and improved sensitivity. Here, we describe a simple procedure to determine the complete complement of intracellular metabolites in cultured malignant cells by HPLC coupled to Q-TOF HRMS. According to this method, (1) cells are collected and processed to minimize contaminations as well as fluctuations in their metabolic profile; (2) samples are separated by HPLC and analyzed on a Q-TOF spectrometer; and (3) data are extracted, normalized, and deconvoluted according to refined mathematical methods. This protocol constitutes a simple approach to determine the intracellular metabolomic profile of cultured cancer cells. With minimal variations (mostly related to sample collection and processing), this method is expected to provide reliable metabolomic data on a variety of cellular samples.
authors.affiliation: Metabolomics and Cell Biology Platform, Gustave Roussy; Villejuif, France; INSERM, U848; Villejuif, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Scoazec
authors.affiliation: Metabolomics and Cell Biology Platform, Gustave Roussy; Villejuif, France; INSERM, U848; Villejuif, France.
authors.forename: Sylvere
authors.initials: S
authors.lastname: Durand
authors.affiliation: Metabolomics and Cell Biology Platform, Gustave Roussy; Villejuif, France; INSERM, U848; Villejuif, France.
authors.forename: Alexis
authors.initials: A
authors.lastname: Chery
authors.affiliation: Université Paris Descartes/Paris V, Sorbonne Paris Cité; Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers; Paris, France; Gustave Roussy; Villejuif, France. Electronic address: deadoc@vodafone.it.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Galluzzi
authors.affiliation: Metabolomics and Cell Biology Platform, Gustave Roussy; Villejuif, France; INSERM, U848; Villejuif, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité; Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers; Paris, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. Electronic address: kroemer@orange.fr.
authors.forename: Guido
authors.initials: G
authors.lastname: Kroemer
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary; D013058:Mass Spectrometry; D055432:Metabolomics; D051379:Mice; D009374:Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D019906:Nuclear Magnetic Resonance, Biomolecular; D014407:Tumor Cells, Cultured
keywords: ATP; Cancer; Glycolysis; HPLC; Metabolomic profiling; Mitochondrial respiration; Q-TOF HRMS