Session: Parallel session 3 - AI and Bioinformatics

Development of a comprehensive labelling strategy metabolomic analysis

 

Metabolomics is a discipline of biology that aims to comprehensively study the metabolites present in a biological sample. These metabolites reflect the metabolic state of an organism at a given moment and can provide valuable insights into various biological processes. They belong to diverse classes of compounds such as lipids, carbohydrates, amino acids, phenols, alkaloids, and terpenes. These different classes of compounds behave very differently in chromatography and mass spectrometry, unlike proteomics, in which all peptides have common analytical properties.

The objective was to develop a methodology for quantitative metabolomics analysis by specifically labeling metabolites bearing the functional groups: OH, NH2, COOH, and C=O. This labeling involves the introduction of a reporter group on the metabolites, which makes them more hydrophobic and enables their identification and quantification. The work encompassed several stages: first, the optimization of labeling protocols for amine and phenol groups of metabolites; then, the development of analytical methods using MALDI-FTICR MS and ESI LC-Orbitrap-MS/MS to identify and quantify these metabolites. Concurrently, a sample normalization method was established to ensure the uniform injection of equivalent quantities of metabolome in each sample.

The results showed that the labeling of amine and phenol functional groups was successful and confirmed with MS/MS on biological samples such as plasma. We managed to eliminate secondary products formed during labeling and established a normalization method by measuring the absorbance at 338 nm and compared to the concentration curve of an amino acid standard mixture. This allowed for the uniform injection of metabolites to compare changes between two or more samples. The calculated coefficient of variation was below 10%, indicating that our method is reproducible. Finally, labelling with reagents specific to carbonyl and carboxylic group is under investigation.