Evaluation of GC/MS-Based 13C-Positional Approaches for TMS Derivatives of Organic and Amino Acids and Application to Plant 13C-Labeled Experiments
2023
Dellero, Younès | Filangi, Olivier | Bouchereau, Alain | Institut de Génétique, Environnement et Protection des Plantes (IGEPP) ; Université de Rennes (UR)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro Rennes Angers ; Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro) | Plateforme de Profilage Métabolique et de Métabolomique (P2M2) ; MetaboHUB-Grand-Ouest ; MetaboHUB-MetaboHUB | IB2021_TRICYCLE of INRAE BAP division | Daniel Monleon | ANR-11-INBS-0010,METABOHUB,Développement d'une infrastructure française distribuée pour la métabolomique dédiée à l'innovation(2011)
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Show more [+] Less [-]English. Analysis of plant metabolite 13C-enrichments with gas-chromatography mass spectrometry (GC/MS) has gained interest recently. By combining multiple fragments of a trimethylsilyl (TMS) derivative, 13C-positional enrichments can be calculated. However, this new approach may suffer from analytical biases depending on the fragments selected for calculation leading to significant errors in the final results. The goal of this study was to provide a framework for the validation of 13C-positional approaches and their application to plants based on some key metabolites (glycine, serine, glutamate, proline, α-alanine and malate). For this purpose, we used tailor-made 13C-PT standards, harboring known carbon isotopologue distributions and 13C-positional enrichments, to evaluate the reliability of GC-MS measurements and positional calculations. Overall, we showed that some mass fragments of proline_2TMS, glutamate_3TMS, malate_3TMS and α-alanine_2TMS had important biases for 13C measurements resulting in significant errors in the computational estimation of 13C-positional enrichments. Nevertheless, we validated a GC/MS-based 13C-positional approach for the following atomic positions: (i) C1 and C2 of glycine_3TMS, (ii) C1, C2 and C3 of serine_3TMS, and (iii) C1 of malate_3TMS and glutamate_3TMS. We successfully applied this approach to plant 13C-labeled experiments for investigating key metabolic fluxes of plant primary metabolism (photorespiration, tricarboxylic acid cycle and phosphoenolpyruvate carboxylase activity).
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