SQ-KFP: A Framework for Spatially Quantitative Metabolic Flux Analysis Enables Imaging the In Vivo Absolute Metabolic...

Alterations in metabolite concentrations often serve as direct drivers of phenotypic variation or disease onset. The changes in metabolite concentrations are directly dependent on the enzymatic reaction rates. However, spatially resolved imaging of the absolute metabolic enzymatic reaction rate (also known as metabolic flux) remains a critical unmet challenge in systems biology. We developed spatially quantitative kinetic flux profiling (SQ-KFP), a framework for quantitatively imaging the metabolic reaction rate, which has the potential for broad application to plant and animal tissues. This approach firstly realized the integration of quantitative flux analysis with mass spectrometry imaging, by generating isotope labeling data with coupled temporal and spatial resolution via image registration. SQ-KFP imaged the backward reaction rate of fumarase (v_FUM) with spatial resolution as a proof-of-concept, revealing different reaction rate densities in petiole-lamina junctions and spatial decoupling between the metabolic reaction rate, metabolite concentration, and isotopic enrichment in leaves of Oxalis corymbosa and Medicago lupulina (validated by various detection methods). These results demonstrate that neither metabolite concentrations nor isotopic labeling values can substitute for reaction rates. Our method provides quantitative imaging of metabolic reaction rates and enables spatial flux analysis across diverse tissues and organisms.