From isotopologues to isotopomers: advances in mass spectrometry-based positional ¹³C analysis in plants

Carbon fluxes throughout metabolic networks can be investigated experimentally through ¹³C-isotope labelling experiments (¹³C-ILE) and ¹³C-metabolic flux analysis (¹³C-MFA). ¹³C-ILE involves the application of isotopically labelled substrates to a biological system to detect label distribution throughout the metabolic network, while ¹³C-MFA is a computational approach that quantitatively estimates fluxes by fitting isotope-labelling data to metabolic network models. ¹³C-MFA can encompass more metabolic reactions and provide subcellular flux estimates that remain difficult to validate directly using current ¹³C-ILE approaches. Overcoming major bottlenecks of ¹³C-ILE is thus needed to improve and validate ¹³C-MFA. Mass spectrometry (MS)-based ¹³C-ILE has been carried out mostly at isotopologue rather than isotopomer level. Isotopologues differ in their isotopic composition, while isotopomers have the same isotopic composition but at different positions within the molecule. Thus, ¹³C-isotopomer approaches discriminate which atoms of a metabolite have received ¹³C, enabling better comprehension of the regulation of metabolic pathways. Here, we highlight the major challenges to improve MS-based positional ¹³C analysis and discuss the advantages of this approach over MS-based methods with low or no carbon-positional resolution. We further demonstrate how positional ¹³C analysis has been used to estimate enzyme activity in vivo and to study plant metabolism.