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.
Highlights
From the ‡Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada; §Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada; ¶Department of Clinical Biochemistry, University Health Network, Toronto, ON M5G 2C4, Canada; ʈDepartment of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada
Our goal here was to develop a straightforward protocol that integrated mammalian cell culture and accurate selected reaction monitoring (SRM) quantification
Concluding Remarks—To summarize, we presented here an SRM-based proteomic workflow to monitor relative expression of proteins in the entire energy metabolic pathway in mammalian cells exposed to different growth conditions
Summary
From the ‡Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada; §Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada; ¶Department of Clinical Biochemistry, University Health Network, Toronto, ON M5G 2C4, Canada; ʈDepartment of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada. The metabolic transformation of cancer cells was studied primarily at the level of genome [6], transcriptome [7], and metabolome [8]. To fully understand the events and outcomes of metabolic transformation of cancer cells, quantitative proteomic approaches are required to complement existing genomic, transcriptomic, and metabolomic approaches. Mass spectrometry-based proteomics, complemented with chemical and metabolic labeling approaches, is a powerful tool for global analysis of protein expression. These approaches have very limited throughput and require extensive sample preparation, complex data analysis, and verification of their results by independent assays. Metabolic labeling is applicable to actively dividing cell lines, but not to the primary cells
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