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Analytical Methods in Targeted Metabolomics and Proteomics Laboratory (TMPL)

TCA/Kreb Cycle Method


Measured Analytes

  1. Alpha ketoglutarate
  2. Citrate
  3. Fumarate
  4. Isocitrate
  5. Lactate
  6. Malate
  7. Methyl malonate
  8. Pyruvate
  9. Succinate
  10. 2-hydroxyglutarate

Chromatography

Liquid chromatography tandem mass spectrometry (LC-MS) was carried out with a Shimadzu Prominence HPLC system and SCIEX 4000 mass spectrometer. An aliquot of 10 µl of each sample was injected onto an Accucore 2.6 µm C18 100 x 2.1 mm column at 40oC for gradient separation. Mobile phases were: A, ddH2O with 0.1% Formic Acid; and B, Acetonitrile with 0.1% Formic Acid. Gradient schedule started at 10% B and was linearly increased to 30% B at 1 minute, 85% B at 5.5 minutes, 95% B at 5.6 minutes and held at 95% B until 6.5 minutes. At 6.5 minutes the gradient was returned to starting conditions and allowed to re-equilibrate until 8.5 minutes. Time per injection was 8.5 minutes.

Sample Processing

  1. Derivatization Method:
    Analyte derivatives for the TCA method were processed as described in Bo Tan et al [1].
    https://www.ncbi.nlm.nih.gov/pubmed/25102203
  2. Extraction Protocols:
    Extraction protocols for sample processing are dependent on the matrices involved. Please see extraction methods section for more specific instructions.

References

[1] Anal Biochem. 2014 Nov 15;465:134-47. doi: 10.1016/j.ab.2014.07.027. Epub 2014 Aug 4.
Derivatization of the tricarboxylic acid intermediates with O-benzyl hydroxylamine for liquid chromatography-tandem mass spectrometry detection.
Tan B, Lu Z, Dong S, Zhao G, Kuo MS.

Resources

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