Mass spectrometry-based methods for advanced metabolic pathway analysis

Background.

Fermentation-based products require carbon and nitrogen sources that compete with the agricultural industry’s needs. Therefore, the use of carbon- and nitrogen-rich waste streams as a substrates and raw materials represents a sustainable supply for advanced biotechnological processes [1]. This shift to microbial biotechnological production requires a deep understanding of metabolic modelling, which will enable the design and development of self-sustaining microbial systems. These systems function independently and robustly through compartmentalization within synthesized subcellular organelles or as a cellular community. As a consequence, there is an increasing demand for small-scale, high-throughput metabolomics studies with the ultimate goal of elucidating efficient microbial metabolic pathways.

Objective.

Develop and implement advanced mass spectrometry (GC-MS) tracer metabolomics methods for the accurate analysis of isotopologues and isotopomers on subcellular fractions and for time-resolved quantification of metabolic flux dynamics.

Methods.

Initially, the project will focus on the collaborative development of rapid and efficient organelle isolation methods targeting mitochondria, microsomes, lysosomes and peroxisomes [2]. In this context, data integrity will be ensured through the design, optimization and validation of the sampling, quenching and storage steps during organelles isolation methods, supported by parallel, small-scale quantitative metabolomics. Gas Chromatography Mass Spectrometry (GC-MS), considered the well-established for accurate measurement of isotopologues and isotopomers, will be the analytical technique of choice for designing fit-for-purpose methods that account for limited sample amounts and time-resolved sampling [3]. Whitin this analytical platform, different configurations will be considered such as GC-single quadrupole, GC-TOF MS and GC-QTOF MS with isotopologues-selective fragmentation [4,5]. Additionally, the project will focus on the development and implementation of a high-throughput platform enabling time-resolved metabolic flux measurements to capture transient metabolic changes that impact on optimizing cell culture processes.

REFERENCES
[1] Ni, C.; Friedman, D. C. Transforming Trash: Strategies to Develop Waste into a Feedstock for a Circular Bioeconomy. Biofuels Bioprod. Biorefining 2024, 18 (5), 1085–1092. https://doi.org/10.1002/bbb.2586.
[2] Satori, C. P.; Kostal, V.; Arriaga, E. A. Review on Recent Advances in the Analysis of Isolated Organelles. Anal. Chim. Acta 2012, 753, 8–18. https://doi.org/10.1016/j.aca.2012.09.041.
[3] Wittmann, C. Fluxome Analysis Using GC-MS. Microb. Cell Factories 2007, 6 (1), 6. https://doi.org/10.1186/1475-2859-6-6.
[4] Mairinger, T.; Hann, S. Implementation of Data-Dependent Isotopologue Fragmentation in 13C-Based Metabolic Flux Analysis. Anal. Bioanal. Chem. 2017, 409 (15), 3713–3718. https://doi.org/10.1007/s00216-017-0339-1.
[5] Mairinger, T.; Steiger, M.; Nocon, J.; Mattanovich, D.; Koellensperger, G.; Hann, S. Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for13 C-Metabolic Flux Analysis. Anal. Chem. 2015, 87 (23), 11792–11802. https://doi.org/10.1021/acs.analchem.5b03173.