Large-Scale Profiling of Cellular Metabolic Activities Using Deep C Labeling Medium.

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 C 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 C 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.