Search

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%.

A mass spectrometric quantitative assay was developed for the analysis of 10 sugar phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten sugar phosphates in yeast extracts utilizing internal standardization with a fully labeled (13)C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.