Benchmarking non-targeted metabolomics using yeast derived libraries

Abstract

Non-targeted analysis by high-resolution mass spectrometry (HRMS) is the essential discovery tool in metabolomics. Up to date, standardization and validation remain a challenge. Community wide accepted, cost-effective benchmark materials are lacking. In this work, we propose yeast (Pichia pastoris) extracts, derived from fully controlled fermentations for this purpose. We established an open-source metabolite library of > 200 metabolites, reproducibly recovered in ethanolic extracts by orthogonal LCHRMS methods, different fermentations (over three years) and different laboratories. More specifically, compound identification was based on accurate mass, matching retention times, and MS/MS spectra as compared to authentic standards and internal databases. The library includes metabolites from the classes of 1) organic acids and derivatives (2) nucleosides, nucleotides and analogues, (3) lipids and lipid-like molecules, (4) organic oxygen compounds, (5) organoheterocyclic compounds, (6) organic nitrogen compounds and (7) benzoids at expected concentrations ranges of sub-nM to µM. As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and all annotated metabolites were also reported in the Human metabolome data base (HMDB). A large fraction of metabolites was found to be stable for several years when stored at −80°C. Thus, the yeast benchmark material enabled not only to test for the chemical space and coverage upon method implementation and developments, but enabled in-house routines for instrumental performance tests. Finally, the benchmark material opens new avenues for batch to batch corrections in large scale non-targeted metabolomics studies.

Recurrent Topics in Mass Spectrometry-Based Metabolomics and Lipidomics—Standardization, Coverage, and Throughput

“…pastoris from fully controlled fermentations, which can be easily reproduced in a lab with fermentor access. 85 Additionally, these extracts are also commercially available in both endogenous and 13 C-labeled formate. An open-source yeast metabolite and lipid library is established for the material.…”

“…Current in-house library of annotated metabolites and lipids found in Pichia pastoris (yeast). (A) Metabolite classes in ethanolic yeast extract classified using the ClassyFirer annotation system. (B) Lipid classes annotated in chloroformic yeast extract .…”

“…The benchmark material is obtained from P . pastoris from fully controlled fermentations, which can be easily reproduced in a lab with fermentor access . Additionally, these extracts are also commercially available in both endogenous and 13 C-labeled formate.…”

“…All reported compounds were reported in the human metabolome data base, showing once more that yeast is a cost-effective benchmark material for human metabolomics. 104 out of 206 metabolites were stable for several years when stored in aliquots at −80 °C …”