Analysis of 13C labeling amino acids by capillary electrophoresis - High resolution mass spectrometry in developing flaxseed.

In context of fluxomic studies, 13C labeling analysis of amino acids are very important for solving the carbon flow calculation, because they are synthesized in various biosynthesis pathways and cellular compartments in plant cells. Traditionally, 13C labeling analysis are performed using low resolution mass spectrometry detector by GC-MS. We compared a method using capillary electrophoresis-high resolution mass spectrometry without derivatization and with better accuracy assessment of labeling measurements comparing to classical GC-MS. Our method allowed us to show that valine, leucine, alanine are not synthesized from the same pyruvate pool during the period of reserves accumulation in flax seeds.

13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].

13C-Metabolic Flux Analysis in Developing Flax (Linum usitatissinum L.) Embryos to Understand Storage Lipid Biosynthesis.

Flax (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3). This polyunsaturated fatty acid is well known for its nutritional role in human and animal diets. Understanding storage lipid biosynthesis in developing flax embryos can lead to an increase in seed yield via marker-assisted selection. While a tremendous amount of work has been done on different plant species to highlight their metabolism during embryo development, a comprehensive analysis of metabolic flux in flax is still lacking. In this context, we have utilized in vitro cultured developing embryos of flax and determined net fluxes by performing three complementary parallel labeling experiments with 13C-labeled glucose and glutamine. Metabolic fluxes were estimated by computer-aided modeling of the central metabolic network including 11 cofactors of 118 reactions of the central metabolism and 12 pseudo-fluxes. A focus on lipid storage biosynthesis and the associated pathways was done in comparison with rapeseed, arabidopsis, maize and sunflower embryos. In our hands, glucose was determined to be the main source of carbon in flax embryos, leading to the conversion of phosphoenolpyruvate to pyruvate. The oxidative pentose phosphate pathway (OPPP) was identified as the producer of NADPH for fatty acid biosynthesis. Overall, the use of 13C-metabolic flux analysis provided new insights into the flax embryo metabolic processes involved in storage lipid biosynthesis. The elucidation of the metabolic network of this important crop plant reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems.

Data documenting the comparison between the theoretically expected values of free sugars mass isotopomer composition with standards using GC-MS and LC-HRMS for Metabolic Flux Analysis.

The data presented in this article are related to the research article entitled "13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux Analysis" (Acket et al., 2017) [1]. This article provides data concerning the comparison between the theoretically expected values of free sugars mass isotopomer composition with standards using our previous methods using low resolution mass spectrometry by GC-MS (Koubaa et al., 2012, 2014) [2,3], and your new method using high resolution-mass spectrometry (LC-HRMS) for Metabolic Flux Analysis [1]. For discussion and a more comprehensive data interpretation and analysis, please refer to Acket et al. (2017) [1].