Isotopomer Analysis Using GC-MS

Christensen, Bjarke Bak; Nielsen, Jens

doi:10.1006/mben.1999.0117

Cited by 216 publications

(190 citation statements)

References 23 publications

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“…The fractional 13 C label of these three pyruvate fragments allows the determination of the abundances of all eight possible 13 C isotopomers in pyruvate, as described by Christensen and Nielsen (1999). Our analysis of pyruvate isotopomers revealed that the relative abundance of [2,3-13 C 2 ]pyruvate was 89% of the abundance defined by sole glycolysis (Fig.…”

Section: Glycolytic Breakdown Of Glc Dominates In Plastidic Acetyl-comentioning

confidence: 66%

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

2002

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Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mm carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mm amino acids as well as 6 to 15 mm malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing 13 C-labeled carbohydrates. The 13 C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of 13 C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of 13 C label into plastid-formed fatty acids, but substantially diluted 13 C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. 13 C label in the terminal acetate units of C 20 and C 22 fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute 13 C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos.Plant oils represent the largest renewable resource of highly reduced carbon chains and there is interest in increasing their production by oilseed crops. Brassica napus (canola, oilseed rape) is a major oil crop and a multitude of literature focuses on the biochemistry and physiology of oil accumulation in developing seeds of B. napus (see Singal et al., 1987;Murphy and Cummis, 1989;Kang and Rawsthorne, 1994;Eastmond and Rawsthorne, 1998;King et al., 1998). Although the biochemical pathways leading from Suc to oil storage are largely understood, a number of questions remain regarding, for example, the subcellular organization of reactions, and the origin of acetyl-CoA, reducing power, and ATP for fatty acid synthesis. These questions are particularly difficult to address using standard in vitro or organellar biochemical analyses that often result in loss of key activities. In addition, due to several reasons including dilution of isotopes by ...

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Section: Glycolytic Breakdown Of Glc Dominates In Plastidic Acetyl-comentioning

confidence: 66%

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

2002

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“…Preliminary information on the network of active biochemical reactions is therefore required. This potential drawback of the described MS technique could be overcome by increasing the number of measured labelings [15]. With regard to a maximized information content from labeling experiments a combination of both, NMR and MS techniques, providing orthogonal information on labeling patterns, may be optimal [15].…”

Section: O N C L U S I O N Smentioning

confidence: 99%

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

Wittmann

Heinzle

2001

European Journal of Biochemistry

113

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In the present work, a novel comprehensive approach of 13 C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 mL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-13 C glucose or with mixtures of 13 C 6 / 12 C 6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.Keywords: MALDI-TOF mass spectrometry; metabolic flux analysis; Corynebacterium glutamicum; lysine; 13 C tracer.Production of amino acids by Corynebacterium glutamicum belongs to the major processes in industrial biotechnology. The world market for amino acids amounts to about 3 billion US dollars, whereby lysine with a worldwide production of about 250 000 tonnes per annum is one of the most important products [1]. Optimization of cultivation strategies and producer strains have led to increased yields and rates of amino-acid production by Corynebacteria. To a large extent, the strain improvement was based on random mutagenesis and subsequent selection [2,3]. In the last decade the powerful tools of genetic engineering allowed the targeted amplification and disruption of selected genes, which led to a number of mutants with increased capabilities of lysine secretion [4]. However, practically achieved yields are still far from theoretical optima, leaving a significant potential for further improvement. To achieve such improvements in a targeted and efficient way, detailed knowledge of intracellular carbon flux distributions and their regulation is needed. Data on genome, transcriptome and proteome ...

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“…Flux determination is thus an essential component of strain evaluation for metabolic engineering (Stephanopoulos, 1999). New methods for flux measurement have been developed based on NMR and MS technologies which allow accurate, high resolution measurements of pathway flux to be made using radiolabeled substrates (Szyperski, 1998;Christensen & Nielsen, 1999). Such methods are based on feeding 13 C-labeled substrates to the cell for the characterization of metabolism (Wiechert, 2001).…”

Section: -Fluxomicsmentioning

confidence: 99%

Wine Science in the Omics Era: The Impact of Systems Biology on the Future of Wine Research

Rossouw

Bauer

2016

SAJEV

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Industrial wine making confronts viticulturalists, wine makers, process engineers and scientists alike with a bewildering array of independent and semi-independent parameters that can in many cases only be optimized by trial and error. Furthermore, as most parameters are outside of individual control, predictability and consistency of the end product remain difficult to achieve. The traditional wine sciences of viticulture and oenology have been accumulating data sets and generating knowledge and know-how that has resulted in a significant optimization of the vine growing and wine making processes. However, much of these processes remain based on empirical and even anecdotal evidence, and only a small part of all the interactions and cause-effect relationships between individual input and output parameters is scientifically well understood. Indeed, the complexity of the process has prevented a deeper understanding of such interactions and causal relationships. New technologies and methods in the biological and chemical sciences, combined with improved tools of multivariate data analysis, open new opportunities to assess the entire vine growing and wine making process from a more holistic perspective. This review outlines the current efforts to use the tools of systems biology in particular to better understand complex industrial processes such as wine making.

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Isotopomer Analysis Using GC-MS

Cited by 216 publications

References 23 publications

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

Wine Science in the Omics Era: The Impact of Systems Biology on the Future of Wine Research

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