Quantifying the extended energy metabolome of industrially important microorganisms (Saccharomyces cerevisiae) using ultra-performance liquid chromatography with mass spectrometry

Abstract

This study has developed a new targeted methodology for the separation, detection, and quantification of metabolites from the wider energy metabolome of industrially important microorganisms such as Saccharomyces cerevisiae in a single analytical sample. This has been achieved using UHPLC-MS technology in HILIC mode. Absolute concentrations of metabolites nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide phosphate reduced (NADPH), flavin adenine dinucleotide (FAD), adenosine-monophosphate (AMP), adenosine-diphosphate (ADP), and adenosine-triphosphate (ATP) were determined in a single extraction and analytical methodology. This study demonstrated the development of a rapid, statistically robust, and reproducible methodology through regression calibrations of standard samples from 0.1 to 100 µMol providing a correlation value of r² = >0.98 for all metabolites. Sample preparation, extraction and analytical methodologies used showed high accuracy, sensitivity, and recovery. With an LOD and LOQ for the targeted analysis of metabolites from the wider energy metabolism in a single sample and analytical run with the lowest LOD of 0.055 nMol (±0.002) and lowest LOQ of 0.167 nMol (±0.006). This method was then applied to Saccharomyces cerevisiae cell culture to evaluate the methodology in industrially used microbial cultures. Results obtained have been statistically determined to be robust and reproducible through recovery analysis using deuterated and isotopically labelled internal standards AMP-¹⁵N, ADP-¹⁵N and ATP-d₁₄.

Keywords: Energy metabolome; Metabolomics; Saccharomyces cerevisiae; Targeted metabolomics; UHPLC-MS.