Positional 13C Enrichment Analysis of Aspartate by GC-MS to Determine PEPC Activity In Vivo

Abstract

Abstract Photoautotrophic organisms fix inorganic carbon (Ci) by two enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC). RUBISCO assimilates Ci (CO2) into the 1-C position of 3-phosphoglycerate (3PGA). The Calvin-Benson-Basham (CBB) cycle redistributes fixed carbon atoms into 2,3-C 2 of the same molecule. PEPC uses phosphoenolpyruvate (PEP) derived from 3PGA and assimilates Ci (HCO 3- ) into 4-C of oxaloacetate (OAA). 1,2,3-C 3 of OAA and of its transaminase product aspartate originate directly from 1,2,3-C 3 of 3PGA. Positional isotopologue analysis of aspartate, the main downstream metabolite of OAA in the model cyanobacterium Synechocystis sp. PCC 6803 ( Synechocystis ), allows differentiation between PEPC, RUBISCO, and CBB cycle activities within one molecule. We explored in source fragmentation of gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) at nominal mass resolution and GC-atmospheric pressure chemical ionization-MS (GC-APCI-MS) at high mass resolution. This enabled the determination of fractional 13 C enrichment (E 13 C) at each carbon position of aspartate. Two prevailing GC-MS derivatization methods, i.e. trimethylsilylation and tert-butyldimethylsilylation, were evaluated. The method was validated by 13 C-isotopomer mixtures of positional labeled aspartic acid. Combination with dynamic 13 CO 2 labeling of Synechocystis cultures allowed direct measurements of PEPC activity in vivo alongside analyses of RUBISCO and CBB cycle activities. Accurate quantification of aspartate concentration and positional E 13 C provided molar Ci assimilation rates during the day and night phases of photoautotrophic Synechocystis cultures. The validated method offers several applications to characterize the photosynthetic Ci fixation in different organisms.