A P2M2 (IGEPP's Metabolic Profiling and Metabolomic Platform) workflow was built based on GC-MS raw data containing multiple ions from each TMS-derivative of interest to: i) correct for natural abundance, ii) calculate carbon isotopologue distributions and mean 13C enrichment, iii) calculate some positional 13C-enrichments. This workflow is specifically dedicated to the analysis of multiple mass fragments from a same metabolite (TMS-derivative) in order to calculate positional 13C enrichments.

The corrective method is publicly available and can be run directly from GC-MS source files using the following workflow :

           Conversion GCMS PostRun Analysis to IsoCor
            Isotopic studies > Isotope Correction for mass spectrometry labeling experiments 
            Calculations of positional 13C-enrichments

The sources are available on the GitHub repository of the P2M2 platform.

The workflow is composed of three items: "Conversion GCMS postrun analysis to IsoCor" , "Isotope Correction for mass spectrometry labeling experiments" and “Calculations of positional 13C-enrichments”.

The first item (gcms2isocor) allows to convert GCMS raw data into a table suitable for IsoCor. For this purpose,the input files must contain a column "Name" filled with each carbon isotopologue of each fragment considered and a column "Area" filled with the area of the integrated peak. The name of each mass fragment analysed in the method file must be written exactly as depicted in the GCMS_example_file . This will ensure that the combination between the chemical formula of the metabolite backbone and the chemical formula of the TMS backbone is correct and clearly identified by IsoCor. Additionally, it will prevent the production of similar “metabolite” with IsoCor arising from different m/z. The identification of chemical structures for each fragment reported in the dataset has been manually curated with respect to known fragmentation patterns and 13C positional studies (see the reference publication). As an example, the name "ProlineC2C5m142_TMS_m0" and "ProlineC2C5m216_2TMSMINUSCH3_m0" are for the GC-MS fragments containing both the C2-C3-C4-C5 carbon skeleton of proline_2TMS derivative but which differed with respect to the m/z monitored (142 or 216) and its subsequently derivative backbone (TMS or 2TMSMINUSCH3).

If this first step of the workflow is bypassed, the users are solely responsible for the scientific credibility of the results generated.

The second item allows to correct for the abundance of naturally occurring isotopes using IsoCor software. Please run IsoCor with the three files “Isotopes.dat”, “Metabolites.dat” and “Derivatives.dat” from the dataset.

The third item allows to calculate all positional 13C-enrichments depending on the fragments contained in the original data. It is possible to edit calculation rules if some fragments are prefered, otherwise all combinations of calculations will be performed. The results can be directly inspected visually and downloaded as a .txt format.