Yeast-based reference materials for quantitative metabolomics

Abstract

We introduce a new concept of yeast-derived biological matrix reference material for metabolomics research relying on in vivo synthesis of a defined biomass, standardized extraction followed by absolute quantification with isotope dilution. The yeast Pichia pastoris was grown using full control- and online monitoring fed-batch fermentations followed by fast cold methanol quenching and boiling ethanol extraction. Dried extracts served for the quantification campaign. A metabolite panel of the evolutionarily conserved primary metabolome (amino acids, nucleotides, organic acids, and metabolites of the central carbon metabolism) was absolutely quantified by isotope dilution utilizing uniformly labeled 13C-yeast-based internal standards. The study involved two independent laboratories employing complementary mass spectrometry platforms, namely hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) and gas chromatography-tandem mass spectrometry (GC–MS/MS). Homogeneity, stability tests (on a panel of >70 metabolites over a period of 6 months), and excellent biological repeatability of independent fermentations over a period of 2 years showed the feasibility of producing biological reference materials on demand. The obtained control ranges proved to be fit for purpose as they were either superior or comparable to the established reference materials in the field.

Achieving Absolute Molar Lipid Concentrations: A Phospholipidomics Cross-Validation Study

“…The better characterization of the K. phaffii yeast lipidome can help to establish a benchmarking tool for the development of an MS-based lipidomics method. , The applied traceable lipid class quantification methods in combination with the simple and reproducible production of K. phaffii fermentation enable a cost-effective and accessible material, which is suitable as a QC system for long-term as well as large-scale studies.…”

“…The better characterization of the K. phaffii yeast lipidome can help to establish a benchmarking tool for the development of an MS-based lipidomics method. 56,57 The applied traceable lipid class quantification methods in combination with the simple and reproducible production of K. phaffii fermentation enable a cost-effective and accessible material, which is suitable as a QC system for long-term as well as large-scale studies. The proposed workflows pave the way for quantitative lipidomics studies including (1) species-unspecific standardization even for new PL classes by 31 P NMR and ICP-MS and (2) support the development of lipid reference materials.…”

An anchored experimental design and meta-analysis approach to address batch effects in large-scale metabolomics

“…In non-model organism experiments, implementation of a BRM 31, 34 also enables stable feature identification. New analytic advances have enabled joint alignment and feature selection across high levels of variability when there is a common QC standard like a BRM included in each batch 77 .…”

“…In metabolomics studies, variation in pre-analytical sample collection (growth), analytical sample preparation (extraction), and data collection (instrument) 31 can be confounded (Figure 1). Identification of shared spectral features using a BRM is a successful strategy 31,32 that has proven essential in large-scale studies [32][33][34] . Implementation of BRM controls for instrument variation can estimate and normalize extraction variation 16,18,31 .…”

Preparation of in-house reference material of Fe, Ca, Ni and Si in carbon sample from an aluminum industry and results of a proficiency test

“…(KOUNBACH et al, 2021). Determining the necessary parameters may be done by an interlaboratory program (WASITO et al,2022).…”