Carbon Isotopomer Analysis with Non-Unifom Sampling HSQC NMR for Cell Extract and Live Cell Metabolomics Studies
Isotopomer analysis using either C-13 NMR or LC/GC-MS has been an invaluable tool for studying metabolic activities in a variety of systems. Traditional challenges are, however, that C-13-detected NMR is insensitive despite its high resolution, and that MS-based techniques cannot easily differentiate positional isotopomers. In addition, current C-13 NMR or LC/GC-MS has limitations in detecting metabolites in living cells. Here, we describe a non-uniform sampling-based 2D heteronuclear single quantum coherence (NUS HSQC) approach to measure metabolic isotopomers in both cell lysates and living cells. The method provides a high resolution that can resolve multiplet structures in the BC dimension while retaining the sensitivity of the H-1-indirect detection. The approach was tested in L1210 mouse leukemia cells labeled with BC acetate by measuring NUS HSQC with 25% sampling density. The results gave a variety of metabolic information such as (1) higher usage of acetate in acetylation pathway than aspartate synthesis, (2) TCA cycle efficiency changes upon the inhibition of mitochondrial oxidative phosphorylation by pharmacological agents, and (3) position-dependent isotopomer patterns in fatty acids in living cells. In addition, we were able to detect fatty acids along with other hydrophilic molecules in one sample of live cells without extraction. Overall, the high sensitivity and resolution along with the application to live cells should make the NUS HSQC approach attractive in studying carbon flux information in metabolic studies.
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