A P2M2 (IGEPP's Metabolic Profiling and Metabolomic Platform) workflow was built based on GC-MS raw data from TBDMS-derivative of metabolites to correct for natural abundance and to calculate carbon isotopologue distributions and mean 13C enrichment using IsoCor2.0 software.
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
The sources are available on the GitHub repository of the P2M2 platform.
The workflow is composed of two items: "Conversion GCMS postrun analysis to IsoCor" and "Isotope Correction for mass spectrometry labeling experiments".
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 TBDMS backbone is correct and clearly identified by IsoCor. The identification of chemical structures for each fragment reported in the dataset has been manually curated with respect to known fragmentation patterns and analysis of 13C-PT standards (MetaboHUB, see the upcoming reference publication). As an example, the name "ProlineC1C5_2TBDMSMINUSC4H9_m0" is for the GC-MS fragment m/z 286 (integrated peak) containing the C2-C3-C4-C5 carbon skeleton of proline_2TBDMS, and m0 refers to the carbon isotopologue monitored (m0 for m/z = 286, m1 for m/z = 287, m2 for m/z = 288, m3 for m/z = 289, m4 for m/z = 290, m5 for m/z = 291). Fragmentation of TBDMS derivatives usually produces two intense ions: [M-57]+ and [M-85]+. The [M-85]+ fragments, corresponding to [M-57]+ fragments with an additional loss of 1 carbon and 1 oxygen, are identified in the metabolite name using "co", i.e. "ProlineC2C5co_2TBDMSMINUSC4H9_m0" for example.
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 (IsoCor from W4M platform) 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.
After this second step, the results can be visualized using custom R scripts or other W4M tools such as "Isoplot: Generate plots from isocor output".
