Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance spectroscopy (NMRS) or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMRS is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring 1% of the sample used for NMRS. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase deficiency in human renal cancers, and investigate the contributions of TCA cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMRS or standard MS to provide outstanding sensitivity in isotope labeling experiments, particularly in vivo. ### Competing Interest Statement R.J.D. is a founder and advisor at Atavistik Bio, and serves on the Scientific Advisory Boards of Agios Pharmaceuticals, Vida Ventures, Droia Ventures and Nirogy Therapeutics. F.C. also works at the National Glycoengineering Research Center at Shandong University.