Large-Scale Profiling of Cellular Metabolic Activities Using Deep 13C Labeling Medium
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The recently developed deep labeling method allows for large-scale profiling of metabolic activities in human cells or tissues using isotope tracing with a highly 13C enriched culture medium in combination with liquid chromatography–high resolution mass spectrometry. This method generates mass spectrometry data sets where endogenous cellular products can be identified, and active pathways can be determined from observed 13C mass isotopomers of the various metabolites measured. Here we describe in detail the experimental procedures for deep labeling experiments in cultured mammalian cells, including synthesis of the deep labeling medium, experimental considerations for cell culture, metabolite extractions and sample preparation, and liquid chromatography–mass spectrometry. We also outline a workflow for the downstream data analysis using publicly available software.
Key wordsCell culture Stable isotope tracing experiments Metabolism Metabolomics LC-HRMS
This work was supported by grants from the Swedish Foundation for Strategic Research (FFL12-0220.006) and Karolinska Institutet to R.N., the National Institutes of Health (NIH) 1R01ES027595 and 1S10OD020025 to M.J., and NIH K01DK116917 to J.D.W.