Measurement uncertainty of isotopologue fractions in fluxomics determined via mass spectrometry

Abstract

Metabolic flux analysis implies mass isotopomer distribution analysis and determination of mass isotopologue fractions (IFs) of proteinogenic amino acids of cell cultures. In this work, for the first time, this type of analysis is comprehensively investigated in terms of measurement uncertainty by calculating and comparing budgets for different mass spectrometric techniques. The calculations addressed amino acids of Pichia pastoris grown on 10% uniformly (13)C labeled glucose. Typically, such experiments revealed an enrichment of (13)C by at least one order of magnitude in all proteinogenic amino acids. Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The samples were diluted to fit the linear dynamic range of the mass spectrometers used (10 μM amino acid concentration). The total combined uncertainties of IFs as well as the major uncertainty contributions affecting the IFs were determined for phenylalanine, which was selected as exemplary model compound. A bottom-up uncertainty propagation was performed according to Quantifying Uncertainty in Analytical Measurement and using the Monte Carlo method by considering all factors leading to an IF, i.e., the process of measurement and the addition of (13)C-glucose. Excellent relative expanded uncertainties (k = 1) of 0.32, 0.75, and 0.96% were obtained for an IF value of 0.7 by LC-MS/MS, GC-MS, and LC-TOFMS, respectively. The major source of uncertainty, with a relative contribution of 20-80% of the total uncertainty, was attributed to the signal intensity (absolute counts) uncertainty calculated according to Poisson counting statistics, regardless which of the mass spectrometry platforms was used. Uncertainty due to measurement repeatability was of importance in LC-MS/MS, showing a relative contribution up to 47% of the total uncertainty, whereas for GC-MS and LC-TOFMS the average contribution was lower (30 and 15%, respectively). Moreover, the IF actually present also depends on the isotopic purity of the carbon sources. Therefore, in the uncertainty calculation a carbon source purity factor was introduced and a minor contribution to the total uncertainty was observed. The results obtained by uncertainty calculation performed according to the Monte Carlo method were in agreement with the uncertainty value of the Kragten approach and showed a Gaussian distribution.

Isotopic Studies of Metabolic Systems by Mass Spectrometry: Using Pascal’s Triangle To Produce Biological Standards with Fully Controlled Labeling Patterns

“…The term "isotopologue" refers to molecular entities that differ only in their isotopic composition (number of isotopic substitutions), according to its IUPAC definition. 11 It consists of isotopic forms that (i) do not have the same isotopic composition but have the same nominal mass (e.g., 13 CH 3 NH 2 and CH 2 DNH 2 ) or (ii) have neither the same isotopic composition nor the same nominal mass (e.g., CH 4 , CH 3 D, CH 2 D 2 ). In the first case, isotopologues can be separated when the MS instrument operates at high mass resolution but cannot be distinguished at a unit mass resolution.…”

“…To validate the proposed strategy for producing metabolites with fully controlled labeling patterns, measured CIDs were compared to predicted CIDs (see Figure 3 and Table S-2 in the Supporting Information). The latter were calculated according to the actual 13 C-enrichment of the labeled methanol substrate, as measured by NMR (50.4%). Because this value is slightly higher than the theoretical 50%, predicted CIDs drifted slightly toward the heaviest isotopologues.…”

Application of Stable Isotope-Assisted Metabolomics for Cell Metabolism Studies

“…The GC-MS is particularly good for studies on central metabolism by measuring amino and organic acids using automated standard library searches. A system evaluation of GC-MS, LC-TOFMS and LC-MS-MS has indicates that low-cost GC-MS with an electron ionization source still provides satisfactory isotopologue analysis of many metabolites and the GC-MS datasets can be directly implemented in fluxomics software [58]. …”

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for the simultaneous determination of l -valine, l -l eucine, l -isoleucine, l -phenylalanine, and l -tyrosine in human serum

“…However, the methods with chemical derivatization suffer from extensive sample preparations as well as long analytical run time . With the development of highly selective and sensitive MS technology, LC–MS/MS has become an acceptable method for the direct analysis of AAs . However, since AAs are high polar and zwitterionic molecules, they almost have no retention on normal C 18 columns, and the chromatographic separation of isomeric AAs such as L‐Leu and L‐Ile were unattainable on normal C 18 columns.…”