Czech Science Foundation, No. 23-06600S (2023-2025)
This project addresses a fundamental analytical limitation of current LC–MS–based metabolomics: the insufficient detectability of chemically unstable metabolites containing aldehyde or keto (oxo) groups. Many biologically essential oxo-metabolites play central roles in energy metabolism, redox balance, and cellular signaling, yet remain analytically inaccessible due to rapid degradation, poor ionization efficiency, or uncontrolled reactions during sample preparation.
The main objective of the project is the basic research and systematic evaluation of novel quaternary aminooxy (QAO) derivatization reagents, including stable-isotope-labeled analogues, designed to stabilize oxo groups and introduce a permanent positive charge. This strategy enables highly sensitive, quantitative LC-MS analysis of more than sixty oxo-metabolites occurring in key metabolic pathways, including the tricarboxylic acid cycle, glycolysis/gluconeogenesis, pentose phosphate pathway, ketone body metabolism, and amino acid metabolism.
The project focuses on three analytically challenging metabolite classes:
(i) oxocarboxylic acids (e.g. pyruvate, oxaloacetate, 2-oxoglutarate),
(ii) reducing sugars and phosphorylated sugar intermediates, and
(iii) reactive aldehydes, including lipid peroxidation products with signaling and pathological relevance.
For each class, new LC-MS workflows are developed that combine in-situ stabilization of oxo groups, isotope-coded derivatization, chromatographic separation, and quantitative MS detection. Particular emphasis is placed on analytical robustness, reaction efficiency, ionization enhancement, and suitability for complex biological matrices and trace-level analysis.
A key strength of the project lies in its mechanistic understanding of electrospray ionization behavior, supported by physico-chemical modeling and systematic evaluation of reagent structure–response relationships. This allows rational optimization of reagent design and provides general principles applicable to future metabolomic method development.
By enabling reliable quantification of previously inaccessible metabolite classes, the project contributes to the methodological foundations of targeted metabolomics. The expected outcomes include new analytical concepts, validated LC-MS workflows, and high-impact publications, with relevance for metabolic research, redox biology, and clinical biochemistry, while remaining firmly grounded in basic analytical chemistry research.
