Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for ¹³C-Metabolic Flux Analysis

Abstract

For the first time an analytical work flow based on accurate mass gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS) with chemical ionization for analysis providing a comprehensive picture of (13)C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for (13)C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, (13)C are central for providing characteristic patterns of labeled metabolites, which in turn give insights into the regulation of metabolic pathway kinetics. The new method enables the analysis of isotopologue fractions of 42 free intracellular metabolites within biotechnological samples, while tandem mass isotopomer information is also accessible for a large number of analytes. Hence, the method outperforms previous approaches in terms of metabolite coverage, while also providing rich isotopomer information for a significant number of key metabolites. Moreover, the established work flow includes novel evaluation routines correcting for isotope interference of naturally distributed elements, which is crucial following derivatization of metabolites. Method validation in terms of trueness, precision, and limits of detection was performed, showing excellent analytical figures of merit with an overall maximum bias of 5.8%, very high precision for isotopologue and tandem mass isotopomer fractions representing >10% of total abundance, and absolute limits of detection in the femtomole range. The suitability of the developed method is demonstrated on a flux experiment of Pichia pastoris employing two different tracers, i.e., 1,6(13)C2-glucose and uniformly labeled (13)C-glucose.

Implementation of data-dependent isotopologue fragmentation in 13C-based metabolic flux analysis

“…This is partly explainable by error propagation, as measurement errors on the respective isotopologue fraction are propagated to the resulting tandem mass isotopomer fractions, since the obtained mass spectral ratio of a fragment on MS2 level is applied to the respective isotopologue fraction.

Fig. 2Isotopologue distribution and tandem mass isotopomer distribution of P. Pastoris cell extract, fed with either (a,b) 50: 50 =  12 C 1 methanol: 13 C 1 methanol or (c,d) U 13 C glucose mixed with natural glucose (20: 80) [12, 17]. Data obtained from the PRM combined with data-dependent fragmentation LC-MS approach, indicated by “LC-QTOFMS AutoMSMS”, is shown in grey, whereas bars in blue depict the obtained fractions from the already published GC-CI-(Q)TOFMS approach.

…”

“…This PRM approach using high mass accuracy mass spectrometry was first employed in the field of quantitative targeted proteomics [15]. We have successfully optimized and implemented this concept to the field of metabolic flux analysis targeting primary metabolites in 13 C MFA using gas chromatography-quadrupole time-of-flight mass spectrometry [12]. In this GC-approach, an isotopologue selective fragmentation using a high-resolution, high mass accuracy GC-QTOFMS instrument was employed for the first time to obtain information on the position of heavy stable isotope by acquiring the full product ion spectrum.…”

A Pareto approach to resolve the conflict between information gain and experimental costs: Multiple-criteria design of carbon labeling experiments

“…For instance, Crown et al propose a precision of 0.4 mol% for their GC-MS setup targeting proteinogenic amino acids [27]. In general, labeling errors depend on the measurement technique, the instrument, the analytic protocols, they can vary between organisms, analytes and the degree of label incorporation [28]. To arrive at realistic error approximations that allow for a fair comparison of the analytical platforms, measurements and their standard deviations were collected from published studies featuring different organisms, platforms and various labeling contents.…”

Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for 13C-MFA

“…Nonetheless, LC-ESI-MS/MS technology is widely used to quantify 13 C-label enrichment in central metabolism intermediates [9, 13–18] and will continue to be used due to some inherent advantages:Cell extracts do not need to be evaporated to dryness in order to perform methoximation or ethoximation and subsequent trimethylsilyl or tert -butyldimethylsilyl derivatization [19–21]. Although the derivatization steps may be automated [22], the total sample processing time in LC-ESI-MS/MS is inherently shorter since cell extracts can be injected directly, at the cost of retention time reproducibility.…”

“…The correction process of GC-MS/MS mass fractions was shown to inflate the coefficient of variation for low intensity (tandem) mass isotopomer fractions [21]. …”