77 research outputs found
Quantification and Mass Isotopomer Profiling of α‑Keto Acids in Central Carbon Metabolism
Mass spectrometry has been established
as a powerful and versatile
technique for studying cellular metabolism. Applications range from
profiling of metabolites to accurate quantification and tracing of
stable isotopes through the biochemical reaction network. Despite
broad coverage of central carbon metabolism, most methods fail to
provide accurate assessments of the α-keto acids oxaloacetic
acid, pyruvate, and glyoxylate because these compounds are highly
reactive and degraded during sample processing and mass spectrometric
measurement. We present a derivatization procedure to chemically stabilize
these compounds readily during quenching of cellular metabolism. Stable
derivatives were analyzed by ultrahigh pressure liquid chromatography
coupled tandem mass spectrometry to accurately quantify the abundance
of α-keto acids in biological matrices. Eventually, we demonstrated
that the developed protocol is suited to measure mass isotopomers
of these α-keto acids in tracer studies with stable isotopes.
In conclusion, the here described method fills one of the last technical
gaps for metabolomics investigations of central carbon metabolism
Absolute range of variation in individual split ratios due to alternate optima for the maximization of biomass yield () and maximization of ATP yield () objectives without additional constraints
<p><b>Copyright information:</b></p><p>Taken from "Systematic evaluation of objective functions for predicting intracellular fluxes in "</p><p>Molecular Systems Biology 2007;3():119-119.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1949037.</p><p>Copyright © 2007, EMBO and Nature Publishing Group</p
Predictive fidelities for aerobic and anaerobic batch cultures in minimal medium with glucose (arbitrary units)
<p><b>Copyright information:</b></p><p>Taken from "Systematic evaluation of objective functions for predicting intracellular fluxes in "</p><p>Molecular Systems Biology 2007;3():119-119.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1949037.</p><p>Copyright © 2007, EMBO and Nature Publishing Group</p> The results were obtained by minimization and maximization of the standardized Euclidean distance of the 10 split ratios for the reference flux solutions (). The four oxygen constraints were not implemented for anaerobic batch cultures. Predictive fidelities above 0.1 are not shown. Crosses and red dots signify that the range of the predictive fidelity is less than 1%. Red dots and bars highlight predictive fidelities without additional constraints. Bars signify the predictive fidelity range for that particular combination of objective function, constraint and environmental condition. For the sole case of nitrate respiration, the upper oxygen uptake rates were translated into corresponding upper bounds for nitrate uptake. Objective functions and constraints are defined in and
Hierarchical cluster trees based on the Euclidean distance among specific agreements ρ for the five objective functions considered in and , including all constraint combinations under the six conditions
<p><b>Copyright information:</b></p><p>Taken from "Systematic evaluation of objective functions for predicting intracellular fluxes in "</p><p>Molecular Systems Biology 2007;3():119-119.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1949037.</p><p>Copyright © 2007, EMBO and Nature Publishing Group</p> Difficult to predict groups of split ratios are highlighted by black lines. Groups of nodes were assigned where the linkage among the nodes was less than 0.7, when the linkage was normalized to values between 0 and 1
Central carbon metabolism of The 10 reactions that describe the actual systemic degree of freedom are indicated in red arrows
<p><b>Copyright information:</b></p><p>Taken from "Systematic evaluation of objective functions for predicting intracellular fluxes in "</p><p>Molecular Systems Biology 2007;3():119-119.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1949037.</p><p>Copyright © 2007, EMBO and Nature Publishing Group</p> These 10 reactions are expressed as 10 split ratios, where each of the 10 reactions that consume a cellular metabolite is divided by the sum of all producing reactions. The corresponding metabolites are indicated in red, whereas the 10 split ratios are shown in blue rectangles. Abbreviations: ACA, acetyl-coenzyme A; ACE, acetate; ACL, acetaldehyd; ACP, acetyl-P; AKG, alpha-ketoglutarate; CIT, citrate; DHP, dihydroxyacetone-P; ETH, ethanol; E4P, erythrose-4-P; FBP, fructose-1,6-bi-P; FOR, formate; FUM, fumarate; F6P, fructose-6-P; GAP, glyceraldehyde-3-P; GLX, glyoxylate; G6P, glucose-6-P; ICT, isocitrate; KDG, 2-keto-3-deoxy-6-phosphogluconate; LAC, lactate; MAL, malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate, 6PG, 6-phosphogluconate; P5P, pentose-5-P; QUH, ubiquinone; QUH2, ubiquinol; S7P, seduheptulose-7-P; SUC, succinate; 3-PG, 3-phosphoglycerate; xt, external
Sensitivity analysis of () the predictive fidelity (arbitrary units) and () acetate secretion on the oxygen uptake constraint in aerobic batch cultures given no assumption on the P-to-O ratio constraint (i
<p><b>Copyright information:</b></p><p>Taken from "Systematic evaluation of objective functions for predicting intracellular fluxes in "</p><p>Molecular Systems Biology 2007;3():119-119.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1949037.</p><p>Copyright © 2007, EMBO and Nature Publishing Group</p>e., in practice a P-to-O ratio of 2 is chosen). The upper bound for the maximal oxygen uptake rate (5–19 mmol/g h) was varied, whereas the minimal experimentally reported maximal oxygen uptake rate (; ; ) is indicated in gray. Blue rectangles are for the maximization of ATP yield, red dots and bars for the maximization of biomass yield and green triangles for the maximization of ATP yield per flux unit. In (A), bars indicate the range of the predictive fidelities, whereas a dot, rectangle or triangle indicates a unique solution. Predictive fidelities above 0.05 are not shown. In (B), the line indicates the experimentally determined value of acetate secretion of 58 mmol/g h ()
Phenotypes of Δ<i>pntAB</i>.
<p>Data are reported as means ± 95% C.I. based on five independent measurements.</p><p><sup>a</sup>No diauxic growth.</p><p>Phenotypes of Δ<i>pntAB</i>.</p
Substrate uptake and secretion rates.
<p>Uptake and secretion rates of glucose (grey) and acetate (white) were determined during growth of <i>E</i>. <i>coli</i> in M9 minimal medium supplemented with either glucose (3 g/l) (A) or glucose (2 g/l) plus sodium acetate (2 g/l) (B). Error bars are 95% C.I. based on three independent measurements.</p
Frequencies of <i>E</i>. <i>coli</i> during growth competition.
<p>SG isolate Z2.2 (A) or its <i>ptsG</i> revertant Z2.2 <i>ptsG</i><sup>WT</sup> (B) were mixed with a FG isolate Z2.4 at various starting ratios and co-cultured in M9 glucose (1 g/l) medium over six passages. Two replicate cultures were performed for each starting ratio. Results from one replicate culture are shown due to good reproducibility.</p
Effects of adaptive mutations on transhydrogenase activity and cAMP concentrations.
<p><i>In vitro</i> enzyme activity of mTH and sTH is shown in the upper panel by grey and white bars, respectively. cAMP concentrations are shown in the lower panel. Mutants whose cAMP concentrations are undetectable (UD) or not determined (ND) are indicated. The detection limit of our cAMP quantification is 10 μM. Dashed lines indicate the mTH and cAMP levels of <i>E</i>. <i>coli</i> ZED. Error bars are 95% C.I. based on three independent measurements.</p
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