title: Monitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-ToF-MS).
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.
authors.affiliation: Center for Systems Biology, University of Iceland, Sturlugata 8, Reykjavik 101, Iceland.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Paglia
authors.forename: Sigrún
authors.initials: S
authors.lastname: Hrafnsdóttir
authors.forename: Manuela
authors.initials: M
authors.lastname: Magnúsdóttir
authors.forename: Ronan M T
authors.initials: RM
authors.lastname: Fleming
authors.forename: Steinunn
authors.initials: S
authors.lastname: Thorlacius
authors.forename: Bernhard Ø
authors.initials: BØ
authors.lastname: Palsson
authors.forename: Ines
authors.initials: I
authors.lastname: Thiele
mesh_terms: D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D001713:Biphenyl Compounds; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000378:metabolism; D011687:Purines / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D015203:Reproducibility of Results; D012265:Ribonucleotides / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology
chemical_list: D001713:Biphenyl Compounds; D011687:Purines; D011743:Pyrimidines; D011753:Pyrones; D012265:Ribonucleotides; D013876:Thiophenes; D000620:Aminoimidazole Carboxamide; C031143:AICA ribonucleotide; C512408:4-hydroxy-3-(4-(2-hydroxyphenyl)phenyl)-6-oxo-7H-thieno(2,3-b)pyridine-5-carbonitrile