title: Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay.
abstract: To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada.
authors.forename: Andrei P
authors.initials: AP
authors.lastname: Drabovich
authors.forename: Maria P
authors.initials: MP
authors.lastname: Pavlou
authors.forename: Apostolos
authors.initials: A
authors.lastname: Dimitromanolakis
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D015687:Cell Hypoxia; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002853:Chromatography, Liquid; D002952:Citric Acid Cycle; D004734:Energy Metabolism; D004958:Estradiol / Q000494:pharmacology; D005690:Galactose / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006019:Glycolysis; D006801:Humans; D016014:Linear Models; D061986:MCF-7 Cells; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D010427:Pentose Phosphate Pathway; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
chemical_list: D010455:Peptides; D020543:Proteome; D005973:Glutamine; D004958:Estradiol; D005947:Glucose; D005690:Galactose