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

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 ¹³C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for ¹³C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, ¹³C 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 <i>Pichia pastoris</i> employing two different tracers, i.e., 1,6¹³C₂-glucose and uniformly labeled ¹³C-glucose

An Interlaboratory Evaluation of Drift Tube Ion Mobility–Mass Spectrometry Collision Cross Section Measurements

Collision cross section (CCS) measurements resulting from ion mobility–mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values (^DTCCS_N2) for over 120 unique ion species with the lowest measurement uncertainty to date. The reproducibility of these ^DTCCS_N2 values are evaluated across three additional laboratories on a commercially available DTIM-MS instrument. The traditional stepped field CCS method performs with a relative standard deviation (RSD) of 0.29% for all ion species across the three additional laboratories. The calibrated single field CCS method, which is compatible with a wide range of chromatographic inlet systems, performs with an average, absolute bias of 0.54% to the standardized stepped field ^DTCCS_N2 values on the reference system. The low RSD and biases observed in this interlaboratory study illustrate the potential of DTIM-MS for providing a molecular identifier for a broad range of discovery based analyses

An Interlaboratory Evaluation of Drift Tube Ion Mobility–Mass Spectrometry Collision Cross Section Measurements

Collision cross section (CCS) measurements resulting from ion mobility–mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values (^DTCCS_N2) for over 120 unique ion species with the lowest measurement uncertainty to date. The reproducibility of these ^DTCCS_N2 values are evaluated across three additional laboratories on a commercially available DTIM-MS instrument. The traditional stepped field CCS method performs with a relative standard deviation (RSD) of 0.29% for all ion species across the three additional laboratories. The calibrated single field CCS method, which is compatible with a wide range of chromatographic inlet systems, performs with an average, absolute bias of 0.54% to the standardized stepped field ^DTCCS_N2 values on the reference system. The low RSD and biases observed in this interlaboratory study illustrate the potential of DTIM-MS for providing a molecular identifier for a broad range of discovery based analyses