This gadget can calculate flux split ratio of the glycolysis (Embden-Meyerhof pathway, EMP) and the pentose phosophate pathway (PPP) based on the labeling data obtained from [1-13C]glucose tracing experiments.

Preparation

Perform tracer experiments using 100% [1-13C]glucose. Other labeled glucose are not supported in this gadget.

Obtain natural isotope-subtracted labeling data by using "Natural Isotope Subtractor".

Procedure

Start Garuda and open the “Split Ratio Calculator”. Choose "Subtracted labeling.csv" generated by "Natural Isotope Subtractor" as input file and click the "Launch" button.

Note:

  • After running "Natural Isotopes Subtractor", the generated data can be transfer smoothly to "Average Labeling Calculator" by clicking the "Discover" button.

    • Select "Mammalian cells" or "Microbial cells" depending on the species to be analyzed.

    Make sure that your experimental conditions meet the conditions indicated in the warning.

    Write an appropriate fragment name depending on the species to be analyzed.

    For mammalian cells

  • Choose a fragment name of 3PG or PEP containing 1st to 3rd carbons. Employment of either pyruvate, alanine, or lactate is not recommeneded due to occurrence of labeling dilution via glutaminolysis.

    • For bacterial cells

  • Choose a fragment names of either alanine, pyruvate, or lactate contatinig 1st to 3rd, and 2nd to 3rd carbons.

    • Note:

  • A split ratio of glycolysis and the pentose phosphate pathway can be calculated based on the following atom mapping. When [1-13C]glucose is metabolized via the glycolysis, the lower glycolytic metabolites are labeled with M : M+1 = 1 : 1, whereas when metabolized via PPP, the first 13C atom of glucose is desorbed as CO2 and the downstream metabolites are unlabeled. Some bacteria has another brach, the Entner-Doudroff pathway. The split ratio of this pathway can be investigated by measring two fragment ions containig 1st to 3rd, and 2nd to 3rd carbons because 13C position of lower glycolytic metabolites is defferent from that produced via glycolysis.

    • A file is generated and it can be saved by double click.

    Note:

  • If the calculated values are negative, a warning message will appear. This is due to errors in 13C-labeling measurements.