Monitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-ToF-MS).

TitleMonitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-ToF-MS).
Publication TypeJournal Article
Year of Publication2012
AuthorsPaglia G, Hrafnsdóttir S, Magnúsdóttir M, Fleming RMT, Thorlacius S, Palsson BØ, Thiele I
JournalAnal Bioanal Chem
Volume402
Issue3
Pagination1183-98
PubMed Date2011-12-14
ISSN1618-2650
KeywordsAminoimidazole Carboxamide, Cell Line, Tumor, Chromatography, High Pressure Liquid, Chromatography, Liquid, Humans, Mass Spectrometry, Metabolomics, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Purines, Pyrimidines, Pyrones, Reproducibility of Results, Ribonucleotides, Thiophenes
Abstract

Here we present an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for extracellular measurements of known and unexpected metabolites in parallel. The method was developed by testing 86 metabolites, including amino acids, organic acids, sugars, purines, pyrimidines, vitamins, and nucleosides, that can be resolved by combining chromatographic and m/z dimensions. Subsequently, a targeted quantitative method was developed for 80 metabolites. The presented method combines a UPLC approach using hydrophilic interaction liquid chromatography (HILIC) and MS detection achieved by a hybrid quadrupole-time of flight (Q-ToF) mass spectrometer. The optimal setup was achieved by evaluating reproducibility and repeatability of the analytical platforms using pooled quality control samples to minimize the drift in instrumental performance over time. Then, the method was validated by analyzing extracellular metabolites from acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) treated with direct (A-769662) and indirect (AICAR) AMP activated kinase (AMPK) activators, monitoring uptake and secretion of the targeted compound over time. This analysis pointed towards a perturbed purine and pyrimidine catabolism upon AICAR treatment. Our data suggest that the method presented can be used for qualitative and quantitative analysis of extracellular metabolites and it is suitable for routine applications such as in vitro drug screening.

Alternate JournalAnal Bioanal Chem
PubMed ID22159369

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