Profiling the Metabolism of Human Cells by Deep ¹³C Labeling

Studying metabolic activities in living cells is crucial for understanding human metabolism, but facile methods for profiling metabolic activities in an unbiased, hypothesis-free manner are still lacking. To address this need, we here introduce the deep-labeling method, which combines a custom ¹³C medium with high-resolution mass spectrometry. A proof-of-principle study on human cancer cells demonstrates that deep labeling can identify hundreds of endogenous metabolites as well as active and inactive pathways. For example, protein and nucleic acids were almost exclusively de novo synthesized, while lipids were partly derived from serum; synthesis of cysteine, carnitine, and creatine was absent, suggesting metabolic dependencies; and branched-chain keto acids (BCKAs) were formed and metabolized to short-chain acylcarnitines, but did not enter the tricarboxylic acid cycle. Remarkably, BCKAs could substitute for essential amino acids to support growth. The deep-labeling method may prove useful to map metabolic phenotypes across a range of cell types and conditions.