Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study
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.
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Elsevier
2015
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Subjects: | Human colon adenocarcinoma HT-29 cells, Metabolomics, Difluoromethylornithine, Polyamines, Capillary electrophoresis-mass spectrometry, |
Online Access: | http://hdl.handle.net/10261/113704 |
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dig-cial-es-10261-1137042018-09-19T10:04:52Z Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study Ibáñez, Clara Simó, Carolina Simó, Carolina Valdés, Alberto Campone, Luca Piccinelli, Anna Lisa García-Cañas, Virginia Cifuentes, Alejandro Ministerio de Educación y Ciencia (España) Human colon adenocarcinoma HT-29 cells Metabolomics Difluoromethylornithine Polyamines Capillary electrophoresis-mass spectrometry 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. This work was supported by an AGL2011-29857-C03-01 project (Ministerio de Educacion y Ciencia, Spain). Peer Reviewed 2015-04-15T11:56:41Z 2015-04-15T11:56:41Z 2015 2015-04-15T11:56:42Z artículo http://purl.org/coar/resource_type/c_6501 doi: 10.1016/j.jpba.2015.03.001 issn: 0731-7085 e-issn: 1873-264X Journal of Pharmaceutical and Biomedical Analysis 110: 83-92 (2015) http://hdl.handle.net/10261/113704 10.1016/j.jpba.2015.03.001 Postprint http://dx.doi.org/10.1016/j.jpba.2015.03.001 Sí open Elsevier |
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Human colon adenocarcinoma HT-29 cells Metabolomics Difluoromethylornithine Polyamines Capillary electrophoresis-mass spectrometry Human colon adenocarcinoma HT-29 cells Metabolomics Difluoromethylornithine Polyamines Capillary electrophoresis-mass spectrometry |
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Human colon adenocarcinoma HT-29 cells Metabolomics Difluoromethylornithine Polyamines Capillary electrophoresis-mass spectrometry Human colon adenocarcinoma HT-29 cells Metabolomics Difluoromethylornithine Polyamines Capillary electrophoresis-mass spectrometry Ibáñez, Clara Simó, Carolina Simó, Carolina Valdés, Alberto Campone, Luca Piccinelli, Anna Lisa García-Cañas, Virginia Cifuentes, Alejandro Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study |
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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. |
author2 |
Ministerio de Educación y Ciencia (España) |
author_facet |
Ministerio de Educación y Ciencia (España) Ibáñez, Clara Simó, Carolina Simó, Carolina Valdés, Alberto Campone, Luca Piccinelli, Anna Lisa García-Cañas, Virginia Cifuentes, Alejandro |
format |
artículo |
topic_facet |
Human colon adenocarcinoma HT-29 cells Metabolomics Difluoromethylornithine Polyamines Capillary electrophoresis-mass spectrometry |
author |
Ibáñez, Clara Simó, Carolina Simó, Carolina Valdés, Alberto Campone, Luca Piccinelli, Anna Lisa García-Cañas, Virginia Cifuentes, Alejandro |
author_sort |
Ibáñez, Clara |
title |
Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study |
title_short |
Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study |
title_full |
Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study |
title_fullStr |
Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study |
title_full_unstemmed |
Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study |
title_sort |
metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: ht-29 cells as case study |
publisher |
Elsevier |
publishDate |
2015 |
url |
http://hdl.handle.net/10261/113704 |
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