Abstract
BackgroundTracing stable isotopes, such as 13C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. However, extracting such information requires special care, such as a correction for naturally occurring isotopes, or overlapping mass spectra of various components of the cell culture medium. Developing a method for a correction of overlapping peaks is the primary objective of this study.ResultsOur computer program-MIDcor (free at https://github.com/seliv55/mid_correct) written in the R programming language, corrects the raw MS spectra both for the naturally occurring isotopes and for the overlapping of peaks corresponding to various substances. To this end, the mass spectra of unlabeled metabolites measured in two media are necessary: in a minimal medium containing only derivatized metabolites and chemicals for derivatization, and in a complete cell incubated medium. The MIDcor program calculates the difference (D) between the theoretical and experimentally measured spectra of metabolites containing only the naturally occurring isotopes. The result of comparison of D in the two media determines a way of deciphering the true spectra. (1) If D in the complete medium is greater than that in the minimal medium in at least one peak, then unchanged D is subtracted from the raw spectra of the labeled metabolite. (2) If D does not depend on the medium, then the spectrum probably overlaps with a derivatized fragment of the same metabolite, and D is modified proportionally to the metabolite labeling. The program automatically reaches a decision regarding the way of correction. For some metabolites/fragments in the case (2) D was found to decrease when the tested substance was 13C labeled, and this isotopic effect also can be corrected automatically, if the user provides a measured spectrum of the substance in which the 13C labeling is known a priori.ConclusionUsing the developed program improves the reliability of stable isotope tracer data analysis.
Highlights
Tracing stable isotopes, such as 13C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells
Gas chromatography coupled to mass spectrometry (GC/MS) is a basic technique used for monitoring 13C isotopic isomers that are different in their mass number
The MIDcor program reveals the distribution of 13C mass isotopomers originating exclusively from artificially 13C enriched substrates
Summary
Tracing stable isotopes, such as 13C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. Methods of the correction for the naturally occurring isotopes 13C and 15N in an analyzed metabolite were first developed by Brauman [10], based on calculations of the theoretical natural MID using the observed frequencies of the isotopes in the environment and chemical compositions of assayed molecules. Overlapping MS signals for more than one metabolite is an important cause of differences between experimentally measured and corresponding MID calculated theoretically We distinguish two such an overlapping cases: either 1) with patterns of unlabeled molecules, which depends only on the composition of the assay medium, or 2) with another pattern of mass isotopomers of the same molecules designated for testing, which depends on their artificial labeling. Our open source software MIDcor (https://github.com/seliv55/ mid_correct), developed in the R programming language, uses the previously described methods for separation of natural and artificial labeling [10, 11, 13], and, corrects the peaks overlapping either with unlabeled or labeled metabolites
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