13C metabolic flux analysis of microbial and mammalian systems is enhanced with GC-MS measurements of glycogen and RNA labeling

Abstract

13C metabolic flux analysis (13C-MFA) is a widely used tool for quantitative analysis of microbial and mammalian metabolism. Until now, 13C-MFA was based mainly on measurements of isotopic labeling of amino acids derived from hydrolyzed biomass proteins and isotopic labeling of extracted intracellular metabolites. Here, we demonstrate that isotopic labeling of glycogen and RNA, measured with gas chromatography-mass spectrometry (GC-MS), provides valuable additional information for 13C-MFA. Specifically, we demonstrate that isotopic labeling of glucose moiety of glycogen and ribose moiety of RNA greatly enhances resolution of metabolic fluxes in the upper part of metabolism; importantly, these measurements allow precise quantification of net and exchange fluxes in the pentose phosphate pathway. To demonstrate the practical importance of these measurements for 13C-MFA, we have used E. coli as a model microbial system and CHO cells as a model mammalian system. Additionally, we have applied this approach to determine metabolic fluxes of glucose and xylose co-utilization in the E. coli ΔptsG mutant. The convenience of measuring glycogen and RNA, which are stable and abundant in microbial and mammalian cells, offers the following key advantages: reduced sample size, no quenching required, no extractions required, and GC-MS can be used instead of more costly LC-MS/MS techniques. Overall, the presented approach for 13C-MFA will have widespread applicability in metabolic engineering and biomedical research.

Fast growth phenotype of E. coli K-12 from adaptive laboratory evolution does not require intracellular flux rewiring

“…For each strain, two parallel labeling experiments were performed with [1,2- 13 C]glucose and [1,6- 13 C]glucose, as this was previously identified to provide optimal precision in flux estimates throughout E. coli central carbon metabolism (Crown et al, 2016). Labeling of proteinogenic amino acids, labeling of ribose moiety of RNA, and glucose moiety of glycogen (Long et al, 2016a) from each parallel experiment were fitted simultaneously, along with the measured acetate yield, to estimate fluxes. The measured mass isotopomer distributions (MID’s) and the estimated metabolic fluxes are provided in Supplemental Materials.…”

“…GC-MS analysis of tert -butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids was performed as described in (Long and Antoniewicz, 2014b). Labeling of glucose (derived from glycogen) and ribose (derived from RNA) were determined as described in (Long et al, 2016a; McConnell and Antoniewicz, 2016). In all cases, mass isotopomer distributions were obtained by integration (Antoniewicz et al, 2007a) and corrected for natural isotope abundances (Fernandez et al, 1996).…”

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis

“…Tracers were purchased from Cambridge Isotope Laboratories: [1,2- 13 C]glucose (99.9 atom% 13 C), [1,6- 13 C]glucose (99.6%), [U- 13 C]glucose (99.3%), [1,2- 13 C]xylose (99.3%), [5- 13 C]xylose (99.6%), and [U- 13 C]xylose (99.3%). The isotopic purity of all glucose tracers was determined by GC-MS (Long et al, 2016a). MOPS minimal medium was used for all experiments.…”

“…Labeling of fatty acids was determined after derivatization to fatty acid methyl esters (FAME) (Crown et al, 2015b). Labeling of glucose (derived from glycogen) and ribose (derived from RNA) were determined as described in (Long et al, 2016a; McConnell and Antoniewicz, 2016). In all cases, mass isotopomer distributions were obtained by integration (Antoniewicz et al, 2007a) and corrected for natural isotope abundances (Fernandez et al, 1996).…”

Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in Escherichia coli

“…Gas chromatography-mass spectrometry (GC-MS) analysis was performed on an Agilent 5977A mass spectrometer to measure labeling of proteinogenic amino acids (48,49), glucose (derived from glycogen), and ribose (from RNA) (26,50,51). Mass isotopomer distributions (MIDs) were obtained by integration and corrected for natural isotope abundances.…”