title: Targeted metabolomics in cultured cells and tissues by mass spectrometry: method development and validation.
abstract: Metabolomics is the identification and quantitation of small bio-molecules (metabolites) in biological samples under various environmental and genetic conditions. Mass spectrometry provides the unique opportunity for targeted identification and quantification of known metabolites by selective reaction monitoring (SRM). However, reproducibility of this approach depends on careful consideration of sample preparation, chemical classes, and stability of metabolites to be evaluated. Herein, we introduce and validate a targeted metabolite profiling workflow for cultured cells and tissues by liquid chromatography-triple quadrupole tandem mass spectrometry. The method requires a one-step extraction of water-soluble metabolites and targeted analysis of central metabolites that include glycolysis, amino acids, nucleotides, citric acid cycle, and the hexosamine biosynthetic pathway. The sensitivity, reproducibility and molecular stability of each targeted metabolite were assessed under experimental conditions. Quantitation of metabolites by peak area ratio was linear with a dilution over a 4 fold dynamic range with minimal deviation R(2)=0.98. Inter- and intra-day precision with cells and tissues had an average coefficient of variation <15% for cultured cell lines, and somewhat higher for mouse liver tissues. The method applied in triplicate measurements readily distinguished immortalized cells from malignant cells, as well as mouse littermates based on their hepatic metabolic profiles.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue R988, Toronto, Ontario M5G 1X5, Canada; Faculty of Pharmacy, Yarmouk University, Irbid, Jordan.
authors.forename: Anas M
authors.initials: AM
authors.lastname: Abdel Rahman
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue R988, Toronto, Ontario M5G 1X5, Canada.
authors.forename: Judy
authors.initials: J
authors.lastname: Pawling
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue R988, Toronto, Ontario M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Ryczko
authors.affiliation: Department of Molecular Genetics, University of Toronto, Canada; The Donnelly Centre, University of Toronto, Canada.
authors.forename: Amy A
authors.initials: AA
authors.lastname: Caudy
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue R988, Toronto, Ontario M5G 1X5, Canada; Faculty of Pharmacy, Yarmouk University, Irbid, Jordan; Department of Molecular Genetics, University of Toronto, Canada; Department of Laboratory Medicine and Pathology, University of Toronto, Canada. Electronic address: dennis@lunenfeld.ca.
authors.forename: James W
authors.initials: JW
authors.lastname: Dennis
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D019343:Citric Acid / Q000032:analysis / Q000378:metabolism; D002952:Citric Acid Cycle; D057809:HEK293 Cells; D006367:HeLa Cells; D006595:Hexosamines / Q000032:analysis / Q000096:biosynthesis; D006801:Humans; D013058:Mass Spectrometry; D055432:Metabolomics / Q000379:methods*; D051379:Mice; D009711:Nucleotides / Q000032:analysis / Q000378:metabolism
chemical_list: D000596:Amino Acids; D006595:Hexosamines; D009711:Nucleotides; D019343:Citric Acid
keywords: Diagnostics; Mass spectrometry; Metabolism; Precision; Sensitivity