Abstract
Metabolic flux analysis is based on the measurement of isotopologue ratios. In this work, a new GC-MS-based method was introduced enabling accurate determination of isotopologue distributions of sugar phosphates in cell extracts. A GC-TOFMS procedure was developed involving a two-step online derivatization (ethoximation followed by trimethylsilylation) offering high mass resolution, high mass accuracy and the potential of retrospective data analysis typical for TOFMS. The information loss due to fragmentation intrinsic for isotopologue analysis by electron ionization could be overcome by chemical ionization with methane. A thorough optimization regarding pressure of the reaction gas, emission current, electron energy and temperature of the ion source was carried out. For a substantial panel of sugar phosphates both of the glycolysis and the pentose phosphate pathway, sensitive determination of the protonated intact molecular ions together with low abundance fragment ions was successfully achieved. The developed method was evaluated for analysis of Pichia pastoris cell extracts. The measured isotopologue ratios were in the range of 55:1-2:1. The comparison of the experimental isotopologue fractions with the theoretical fractions was excellent, revealing a maximum bias of 4.6% and an average bias of 1.4%.
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