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Title

Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study

AuthorsIbáñez, Clara ; Simó, Carolina ; Simó, Carolina ; Valdés, Alberto ; Campone, Luca; Piccinelli, Anna Lisa; García-Cañas, Virginia ; Cifuentes, Alejandro
KeywordsHuman colon adenocarcinoma HT-29 cells
Metabolomics
Difluoromethylornithine
Polyamines
Capillary electrophoresis-mass spectrometry
Issue Date2015
PublisherElsevier
CitationJournal of Pharmaceutical and Biomedical Analysis 110: 83-92 (2015)
AbstractIn 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.
Publisher version (URL)http://dx.doi.org/10.1016/j.jpba.2015.03.001
URIhttp://hdl.handle.net/10261/113704
DOI10.1016/j.jpba.2015.03.001
Identifiersdoi: 10.1016/j.jpba.2015.03.001
issn: 0731-7085
e-issn: 1873-264X
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