Positional¹³C Enrichment Analysis of Aspartate by GC-MS to Determine PEPC ActivityIn Vivo

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-C2 of the same molecule. PEPC uses phosphoenolpyruvate (PEP) derived from 3PGA and assimilates Ci (HCO3-) into 4-C of oxaloacetate (OAA). 1,2,3-C3 of OAA and of its transaminase product aspartate originate directly from 1,2,3-C3 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 13C enrichment (E13C) 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 13C-isotopomer mixtures of positional labeled aspartic acid. Combination with dynamic 13CO2 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 E13C 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.