Quantifying intracellular metabolites in yeast using a matrix with minimal interference from naturally occurring...

Area of Science:

• Metabolomics
• Analytical Chemistry
• Biochemistry

Background:

• Accurate quantification of intracellular metabolites is crucial for understanding cellular processes.
• Current methods often require complex validation due to metabolite interference in biological matrices.
• Stable isotope-labeled internal standards (SIL-ISs) are essential for precise mass spectrometry-based quantification.

Purpose of the Study:

• To develop an alternative validation strategy for intracellular metabolite quantification.
• To create a biological matrix with minimal signal interferences for analytes and their SIL-ISs.
• To apply the developed methodology for quantifying various metabolites in Saccharomyces cerevisiae.

Main Methods:

• Cultivation of Saccharomyces cerevisiae in a defined growth medium containing both (13)C6-labeled and nonlabeled glucose.
• Preparation of a biological matrix from yeast cultures to minimize signal interferences.
• Validation of the matrix using standard addition for spiking nonlabeled and SIL-ISs.
• Quantification of nucleotides, coenzymes, and redox compounds using mass spectrometry techniques.
• Optimization of sample preparation for sensitive redox compounds like NADH and NADPH.

Main Results:

• A novel matrix was produced from Saccharomyces cerevisiae, showing minimal signal interference (ATP isotope fractions at 2% of total).
• The matrix facilitated straightforward validation with spiking of nonlabeled and SIL-ISs, achieving intra- and inter-day accuracy (⩾80%) and precision (⩽20%).
• Quantification of energy charge ratio (0.9) and Mal-CoA/Ac-CoA ratio (0.04) was achieved, alongside analysis of redox compounds.
• Dissolving redox compounds in ammonium acetate solution (pH 8.0) mitigated oxidation issues.

Conclusions:

• The developed matrix provides a robust and simplified approach for validating intracellular metabolite quantification.
• This methodology enables accurate and precise analysis of diverse metabolites, including energy compounds and redox cofactors, in Saccharomyces cerevisiae.
• The study highlights the importance of optimized sample preparation for challenging analytes like NADH and NADPH in metabolomic studies.