FluxFix: automatic isotopologue normalization for metabolic tracer analysis

Abstract

Background: Isotopic tracer analysis by mass spectrometry is a core technique for the study of metabolism. Isotopically labeled atoms from substrates, such as [¹³C]-labeled glucose, can be traced by their incorporation over time into specific metabolic products. Mass spectrometry is often used for the detection and differentiation of the isotopologues of each metabolite of interest. For meaningful interpretation, mass spectrometry data from metabolic tracer experiments must be corrected to account for the naturally occurring isotopologue distribution. The calculations required for this correction are time consuming and error prone and existing programs are often platform specific, non-intuitive, commercially licensed and/or limited in accuracy by using theoretical isotopologue distributions, which are prone to artifacts from noise or unresolved interfering signals.

Results: Here we present FluxFix ( http://fluxfix.science ), an application freely available on the internet that quickly and reliably transforms signal intensity values into percent mole enrichment for each isotopologue measured. 'Unlabeled' data, representing the measured natural isotopologue distribution for a chosen analyte, is entered by the user. This data is used to generate a correction matrix according to a well-established algorithm. The correction matrix is applied to labeled data, also entered by the user, thus generating the corrected output data. FluxFix is compatible with direct copy and paste from spreadsheet applications including Excel (Microsoft) and Google sheets and automatically adjusts to account for input data dimensions. The program is simple, easy to use, agnostic to the mass spectrometry platform, generalizable to known or unknown metabolites, and can take input data from either a theoretical natural isotopologue distribution or an experimentally measured one.

Conclusions: Our freely available web-based calculator, FluxFix ( http://fluxfix.science ), quickly and reliably corrects metabolic tracer data for natural isotopologue abundance enabling faster, more robust and easily accessible data analysis.

Keywords: Correction; Enrichment; Flux; Isotopologue; Metabolite; Normalization; Tracer.

Fig. 1

Incorporation of ¹³C-labeled substrate can be measured by mass changes in product metabolites. U-[¹³C₆]-glucose incorporation into acetyl-CoA and subsequently into HMG-CoA is shown here as an example. Carbons derived from glucose can be incorporated into acetyl-CoA, and subsequently into HMG-CoA in units of 2. Thus, 2, 4 or 6 labeled carbons can be added to a HMG-CoA molecule, producing the M2, M4 or M6 isotopologues, respectively

Fig. 2

Molecular structure of acetyl-CoA and HMG-CoA. Carbon from glucose can be incorporated into the R-groups. The MS2 fragment measured experimentally incorporates the R-groups, as well as 11 other carbon molecules. Carbon atoms are highlighted as red circles

Fig. 3

Data correction for acetyl-CoA and HMG-CoA using FluxFix. Input data as signal intensity (left y-axis) are in black and grey and output percent molar enrichment data (right y-axis) are in red. Molar enrichment from [¹³C]-glucose occurs in the M2 for acetyl-CoA and M2, M4 and M6 isotopologues for HMG-CoA. This incorporation of glucose is consistent with the known metabolic pathways by which glucose carbon is incorporated in pairs and to a maximum of two atoms for acetyl-CoA and six atoms for HMG-CoA. Data is from three replicate samples, error bars are standard deviation