Yeast-based reference materials for quantitative metabolomics

Hendri Wasito,Gerrit Hermann,Veronika Fitz,Stephan Hann,Gunda Koellensperger,Christina Troyer

doi:10.1007/s00216-021-03694-w

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.

Highlights

The emergence of large-scale metabolomics screenings in clinical research and other regulated environments calls for harmonization
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
We investigated an alternative route of producing biological reference materials

Summary

Introduction

The emergence of large-scale metabolomics screenings in clinical research and other regulated environments calls for harmonization. Definitions for minimum quality requirements together with harmonized protocols for measurement and reporting are established. Quantitative measurements require validation schemes integrating standards and reference materials. In -omics type of analysis, this concept is gaining significant momentum; the pace of developing routine applications is slow due to the lack and the costs of standards [7]. For large-scale metabolomics and lipidomics studies, the use of pooled samples is proposed as a minimum requirement improving intra- and inter-batch repeatability [8, 9]. The integration of multi-standard panels in ready to use, well-plate formats proofed to be a valuable strategy [7]. Interlaboratory comparisons and the wide adoption of kit-type analysis advanced harmonization

Methods

Results

Conclusion