title: Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study.
abstract: In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed.
authors.affiliation: Laboratory of Foodomics, Institute of Food Science Research (CIAL), CSIC, Nicolas Cabrera 9, Cantoblanco Campus, 28049 Madrid, Spain. Electronic address: clara.ibanez@csic.es.
authors.forename: Clara
authors.initials: C
authors.lastname: Ibáñez
authors.affiliation: Laboratory of Foodomics, Institute of Food Science Research (CIAL), CSIC, Nicolas Cabrera 9, Cantoblanco Campus, 28049 Madrid, Spain. Electronic address: c.simo@csic.es.
authors.forename: Carolina
authors.initials: C
authors.lastname: Simó
authors.affiliation: Laboratory of Foodomics, Institute of Food Science Research (CIAL), CSIC, Nicolas Cabrera 9, Cantoblanco Campus, 28049 Madrid, Spain. Electronic address: a.valdes@csic.es.
authors.forename: Alberto
authors.initials: A
authors.lastname: Valdés
authors.affiliation: Dipartimento di Farmacia, University of Salerno, Via Giovanni Paolo II 132, 84084, Fisciano (SA), Italy. Electronic address: lcampone@unisa.it.
authors.forename: Luca
authors.initials: L
authors.lastname: Campone
authors.affiliation: Dipartimento di Farmacia, University of Salerno, Via Giovanni Paolo II 132, 84084, Fisciano (SA), Italy. Electronic address: apiccine@unisa.it.
authors.forename: Anna Lisa
authors.initials: AL
authors.lastname: Piccinelli
authors.affiliation: Laboratory of Foodomics, Institute of Food Science Research (CIAL), CSIC, Nicolas Cabrera 9, Cantoblanco Campus, 28049 Madrid, Spain. Electronic address: virginia.garcia@csic.es.
authors.forename: Virginia
authors.initials: V
authors.lastname: García-Cañas
authors.affiliation: Laboratory of Foodomics, Institute of Food Science Research (CIAL), CSIC, Nicolas Cabrera 9, Cantoblanco Campus, 28049 Madrid, Spain. Electronic address: a.cifuentes@csic.es.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Cifuentes
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D000970:Antineoplastic Agents / Q000494:pharmacology; D002448:Cell Adhesion; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D000518:Eflornithine / Q000494:pharmacology; D019075:Electrophoresis, Capillary; D005240:Feasibility Studies; D019073:HT29 Cells; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D065108:Ornithine Decarboxylase Inhibitors / Q000494:pharmacology; D011073:Polyamines / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000970:Antineoplastic Agents; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D000518:Eflornithine
keywords: Capillary electrophoresis-mass spectrometry; Difluoromethylornithine; Human colon adenocarcinoma HT-29 cells; Metabolomics; Polyamines