title: GC-TOFMS analysis of metabolites in adherent MDCK cells and a novel strategy for identifying intracellular metabolic markers for use as cell amount indicators in data normalization.
abstract: Cultured cell lines are useful models in biomedical research that characterize metabolic responses to various stimuli (e.g., pathogens, toxins, or drugs/chemicals) and explore the underlying mechanisms. However, data from cell metabolomic studies must be normalized to the amount of cells, which is dependent on diverse treatments. The currently used methods of cell counting and protein assay involve extra work and delay the quenching of intracellular metabolism. To develop a convenient, alternative approach, in this study, intracellular metabolites were extracted from a series amount of cultured adherent cells and profiled by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The GC-TOFMS signal intensities for 11 intracellular markers present in two different cell lines showed good linearity with the protein content, with inositol and pantothenate most promising (correlation coefficient > 0.970). Despite the various amounts of cells, the data normalized to the metabolic markers and protein amounts showed similar effectiveness, resulted in better separation of the two cell lines, closer clustering within each group(cell line) on a principal components analysis scores plot, and had lower relative standard deviations for intracellular metabolites than those of the non-normalized data, suggesting that these markers were effective indicators of cell amounts and independent of cell lines.
authors.affiliation: Laboratory of Metabolomics, China Pharmaceutical University, Nanjing, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Cao
authors.forename: Jiye
authors.initials: J
authors.lastname: Aa
authors.forename: Guangji
authors.initials: G
authors.lastname: Wang
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Wu
authors.forename: Linsheng
authors.initials: L
authors.lastname: Liu
authors.forename: Mengjie
authors.initials: M
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Shi
authors.forename: Xinwen
authors.initials: X
authors.lastname: Wang
authors.forename: Chunyan
authors.initials: C
authors.lastname: Zhao
authors.forename: Tian
authors.initials: T
authors.lastname: Zheng
authors.forename: Sheng
authors.initials: S
authors.lastname: Guo
authors.forename: Jinao
authors.initials: J
authors.lastname: Duan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D004285:Dogs; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1