Quantitative metabolome analysis using liquid chromatography-high-resolution mass spectrometry.

Area of Science:

• Analytical Chemistry
• Metabolomics
• Biochemistry

Background:

• Metabolome quantification is crucial for understanding cellular processes.
• High-resolution mass spectrometry offers enhanced sensitivity and specificity for metabolite analysis.

Purpose of the Study:

• To develop and validate a liquid chromatography-single mass spectrometry method for accurate metabolome quantification.
• To assess the method's performance in complex biological matrices.
• To investigate metabolic profiles of Methylobacterium extorquens AM1 under different growth conditions.

Main Methods:

• Hydrophilic interaction liquid chromatography (HILIC) coupled with LTQ Orbitrap high-resolution mass spectrometry.
• Isotope dilution method for accurate quantification in complex samples.
• Analysis of intracellular metabolite concentrations in Methylobacterium extorquens AM1.

Main Results:

• The method achieved limits of detection from 50 fmol to 5 pmol for 25 metabolites.
• Excellent linearity (R(2)>0.99) was observed over 2-3 orders of magnitude.
• Accurate quantification was achieved in complex samples with metabolite concentrations ranging from 0.13 to 55.6 µM.
• Significant variations in intracellular metabolite pool sizes were identified between methanol and succinate growth conditions.

Conclusions:

• The developed LC-MS method provides a sensitive and accurate approach for metabolome quantification.
• The LTQ Orbitrap system offers new strategies for high-resolution metabolomics.
• Metabolite profiles of M. extorquens AM1 are significantly influenced by carbon source availability.