Increasing the accuracy of mass isotopomer analysis through calibration curves constructed using biologically synthesized compounds

Abstract

Mass isotopomer analysis is an important technique to measure the production and flow of metabolites in living cells, tissues, and organisms. This technique depends on accurate quantifications of different mass isotopomers using mass spectrometry. Constructing calibration curves using standard samples is the most universal approach to convert raw mass spectrometry measurements into quantitative distributions of mass isotopomers. Calibration curve approach has been, however, of very limited use in comprehensive analyses of biological systems, mainly suffering from the lack of extensive range of standard samples with accurately known isotopic enrichment. Here, we present a biological method capable of synthesizing specifically labeled amino acids. These amino acids have well-determined and estimable mass isotopomer distributions and thus can serve as standard samples. In this method, the bacterium strain Methylobacterium salsuginis sp. nov. was cultivated with partially 13 C-labeled methanol as the only carbon source to produce 13 C-enriched compounds. We show that the mass isotopomer distributions of the various biosynthesized amino acids are well determined and can be reasonably estimated based on proposed binomial approximation if the labeling state of the biomass reached an isotopic steady state. The interference of intramolecular inhomogeneity of 13 C isotope abundances caused by biological isotope fractionation was eliminated by estimating average 13 C isotope abundance. Further, the predictions are tested experimentally by mass spectrometry (MS) spectra of the labeled glycine, alanine, and aspartic acid. Most of the error in mass spectrometry measurements was less than 0.74 mol% in the test case, significantly reduced as compared with uncalibrated results, and this error is expected to be less than 0.4 mol% in real experiment as revealed by theoretical analysis.

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

“…Isotopic fractionation associated with enzymatic activities is reported in the literature, although, in 13 C-labeling experiments, such effects are considered to be negligible. 1,25,29 The data presented here are consistent with this expectation. They indicate that isotopic effects, if any, are lower than the precision of CID measurements.…”

A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress

“…Assuming that the labeling biomass follows first-order wash-out kinetics, the GC-MS and NMR data of the amino acid isotopomer composition (Additional file 1) were corrected for the deviation from the isotopic steady state at the time of harvesting [20,55,56], using an isotopic nonsteady-state correction [57,58]. …”

Determination of isotopic labeling of proteins by precursor ion scanning liquid chromatography/tandem mass spectrometry of derivatized amino acids applied to nuclear magnetic resonance studies

“…IRMS experiments yield very accurate results for isotope measurements; however, they do not allow any structural information to be extracted from the data since all molecules are transformed into combustion products prior to entering the ion source, also necessitating highly specialized instrumentation, and, furthermore, would not be easily amenable for simultaneous determination of 15 N enrichment. GC/MS is a common technique used for isotope analysis and has often been applied to the study of amino acid isotopic enrichment . Most GC/MS analyses use time‐consuming derivatization steps in order to transform analytes into volatile species and resulting electron ionization (EI) spectra are often very difficult to interpret when many isotopomers are present at once .…”

“…GC/MS is a common technique used for isotope analysis and has often been applied to the study of amino acid isotopic enrichment. [6][7][8][9] Most GC/MS analyses use time-consuming derivatization steps in order to transform analytes into volatile species and resulting electron ionization (EI) spectra are often very difficult to interpret when many isotopomers are present at once. [10] LC/MS for isotope analysis of amino acids has previously been reported, demonstrating the power and applicability of this technique.…”