Determination of Complex Isotopomer Patterns in Isotopically Labeled Compounds by Mass Spectrometry

Abstract

A classic problem in analytical chemistry has been determination of individual components in a mixture without availability of the pure individual components. Measurement of the distribution of isotopomers in a labeled compound or mixture of labeled compounds is an example of this problem that is commonly encountered when stable isotopically labeled metabolites are used to determine in vivo kinetics and metabolism. We present a method that uses the measured mass spectral data of the unlabeled material to represent any and all combinations of isotopomer variations of that material and to determine abundances of these isotopomers. Although examples of the method are presented for gas chromatography-mass spectrometry, the method is applicable to any type of mass spectrometry data. The method also accounts for errors induced by mass spectrometer ionization and resolution effects. To demonstrate this method, we determined the isotopomer distributions of samples of 13 C-labeled leucine and glucose for both highly enriched isotopomers and labeled isotopomers present in low abundance against a natural isotopic abundance background. The method accurately and precisely determined isotopomer identity and abundance in the labeled materials without adding noise or error that was not inherent in the original mass spectral data. In examples shown here isotopomer uncertainties were calculated with relative standard errors of <1% from good quality mass spectral data.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies

“…Isotopomer analysis of the intracellular and extracellular TCA cycle metabolites from the HMCL cell pellets and spent media, respectively, were performed using an Agilent Technologies 5975C GC-MS. Splitless injections of 1-μl aliquots of the derivatized extracts were injected onto a DB5-MS capillary column ( . The mass isotopomer distribution of each compound was then corrected for natural abundance using the respective standards (30). We used an appropriate set of linear simultaneous equations to calculate mole percent enrichment of TCA cycle intermediates to understand glucose-dependent or glucose-independent glutamine metabolism in myeloma cells.…”

Non-targeted Tracer Fate Detection

“…Jennings and Matthews [27] presented an elegant method to correct raw MIDs for natural isotope abundance through comparison with the mass spectrum of the unlabeled compound. An adaptation of this approach is applied in NTFD.…”

“…Depending of the natural isotope abundance of the isotopic element, a M 2 correction may become necessary as described in Jennings and Matthews [27]. Combined labeling from different isotopic elements (e.g.…”

Quantitative Determination of Isotope Ratios from Experimental Isotopic Distributions

“…[18,19] Progress has also been made for the measurement of the distribution of isotopomers in a labeled compound by means of linear algebra. [20] However, the purpose of the current approach is to determine the natural abundances of elements from their experimental isotopic distributions. This work should not be confused with the approaches intended for monitoring the progress of isotope enrichment ratios used in isotope labeling experiments.…”