Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for ¹³C‑Metabolic Flux Analysis

For the first time an analytical work flow based on accurate mass gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS) with chemical ionization for analysis providing a comprehensive picture of ¹³C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for ¹³C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, ¹³C are central for providing characteristic patterns of labeled metabolites, which in turn give insights into the regulation of metabolic pathway kinetics. The new method enables the analysis of isotopologue fractions of 42 free intracellular metabolites within biotechnological samples, while tandem mass isotopomer information is also accessible for a large number of analytes. Hence, the method outperforms previous approaches in terms of metabolite coverage, while also providing rich isotopomer information for a significant number of key metabolites. Moreover, the established work flow includes novel evaluation routines correcting for isotope interference of naturally distributed elements, which is crucial following derivatization of metabolites. Method validation in terms of trueness, precision, and limits of detection was performed, showing excellent analytical figures of merit with an overall maximum bias of 5.8%, very high precision for isotopologue and tandem mass isotopomer fractions representing >10% of total abundance, and absolute limits of detection in the femtomole range. The suitability of the developed method is demonstrated on a flux experiment of <i>Pichia pastoris</i> employing two different tracers, i.e., 1,6¹³C₂-glucose and uniformly labeled ¹³C-glucose

Additional file 3: of Systems-level organization of yeast methylotrophic lifestyle

Enrichment of the peroxisomal marker protein Pex3p in the peroxisomal fraction. (PDF 271 kb

Additional file 1: of Systems-level organization of yeast methylotrophic lifestyle

Transcriptomic, proteomic, and metabolomic regulation of P. pastoris during methylotrophic growth. Containing the following eight sheets: Summary Omics Data: number of significantly regulated genes, proteins or metabolites (e.g. “up” refers to up-regulation in methanol/glycerol compared to glucose). Transcriptomics and proteomics: Average fold changes and P values of transcriptomics and proteomics comparing P. pastoris cultivated with methanol/glycerol or glucose as carbon source in chemostat. Average values derive from three biological replicates per condition. Metabolomics: Average fold changes and P values of metabolomics measurements comparing P. pastoris cultivated with methanol/glycerol or glucose as carbon source in chemostat cultivations. Average values derive from three biological replicates per condition. Co-regulation (related to Fig. 1 in the text): Regulation of the 575 gene-protein pairs with transcriptomics and proteomics data available and assignment to regulatory groups. Central carbon metabolism (related to Fig. 4 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 4. Amino acid metabolism (related to Fig. 6 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 6. Vitamin biosynthesis (related to Fig. 7 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 7. Peroxisomal gene regulation: Average fold changes and P values of transcriptomics and proteomics for all mentioned peroxisomal genes. Average values derive from three biological replicates per condition. (XLSX 2348 kb

Additional file 4: of Systems-level organization of yeast methylotrophic lifestyle

Proteomic identification and quantification of methanol metabolic enzymes and control proteins in peroxisomal fractions and homogenates of P. pastoris cells grown on methanol. Containing the following three sheets: Protein hits: contains all identified proteins that met the threshold in at least one sample, with their respective MASCOT scores, number of peptides, and percent sequence coverage. Peptide hits: list of all identified peptides, their MASCOT scores, mass and charge values, and intensities. Peptides used for quant + areas: lists all peptides of the proteins in Table 3 that were used for quantification, and their respective peak areas in the different samples. (XLSX 879 kb