Quantifying the extended energy metabolome of industrially important microorganisms (Saccharomyces cerevisiae) using ultra-performance liquid chromatography with mass spectrometry
Year: 2024
Type: article
Abstract: • A relatively simple, fast, and accurate methodology for the determination of nucleotide concentrations from the wider energy metabolome of Saccharomyces cerevisiae using a zwitterionic column and UHPLC-MS instrumentation was developed and robustly validated. This is, to the best of our knowledge, the only methodology that does not require samples to be pre-spiked with the metabolites intended for detection and quantification. • This method relies upon the polar chemically bonded internal chemistry of the BEH HILIC column for the detection, separation, and quantification of polar to highly polar metabolites, within one methodology. Furthermore, this method used adapted established quenching and extraction protocols for the processing of Saccharomyces cerevisiae cell cultures. • The major advantages of this methodology include the runtime of 10 min per sample, as well as the use of a single triple quadrupole MS detection unit, which is standard equipment in many laboratories, and the use of widely available labelled isotopic or deuterated internal standards. 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 r 2 = >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- 15 N, ADP- 15 N and ATP-d 14 .
Source: Journal of Chromatography B
Authors: Jordan I. Oliver, Antony N. Davies, Richard Dinsdale
Institution: University of South Wales
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Award: G4362600037
OpenAlex ID: W4403711807