Isotope labeling pattern study of central carbon metabolites using GC/MS

Area of Science:

• Metabolic Engineering
• Systems Biology
• Analytical Chemistry

Background:

• Metabolic flux analysis relies on tracking (13)C tracer labeling patterns in metabolites.
• Liquid chromatography-mass spectrometry (LC/MS) is commonly used but has limitations.
• Gas chromatography-mass spectrometry (GC/MS) offers advantages in sensitivity, cost, and ease of use for isotope pattern studies.

Purpose of the Study:

• To demonstrate the analysis of isotope patterns for central carbon metabolites using GC/MS.
• To quantify isotope fractions and monitor dynamic changes in metabolic intermediates.
• To establish GC/MS as a viable platform for metabolome-based (13)C metabolic flux analysis.

Main Methods:

• Selection of 34 mass fragment ions for 25 central carbon metabolites based on carbon backbone and isomer information.
• Construction of a natural mass isotopomer library for subtraction from experimental data.
• Monitoring dynamic changes in isotope fragments using a U-(13)C glucose stimulus response experiment in Kluyveromyces marxianus.

Main Results:

• Successful quantification of isotope patterns and fractions for 25 central carbon metabolites.
• Discovery of high abundance of partially labeled (13)C intermediates, e.g., fructose 6-phosphate (56.4%) and dihydroxyacetone phosphate (47.6%), generated in the pentose phosphate pathway.
• Demonstration of dynamic changes in isotope fragments under stimulus response.

Conclusions:

• GC/MS is a powerful and practical tool for analyzing (13)C labeling patterns in central carbon metabolism.
• The method allows for precise quantification of isotopic fractions and dynamic flux analysis.
• Accurate isotope pattern acquisition using GC/MS is crucial for robust metabolic flux calculations.