Yeast-based reference materials for quantitative metabolomics.

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.

Alternating in-source fragmentation with single-stage high-resolution mass spectrometry with high annotation confidence in non-targeted metabolomics.

Non-targeted metabolomics is increasingly applied in various applications for understanding biological processes and finding novel biomarkers in living organisms. However, high-confidence identity confirmation of metabolites in complex biological samples is still a significant bottleneck, especially when using single-stage mass analysers. In the current study, a complete workflow for alternating in-source fragmentation on a time-of-flight mass spectrometry (TOFMS) instrument for non-targeted metabolomics is presented. Hydrophilic interaction liquid chromatography (HILIC) was employed to assess polar metabolites in yeast following ESI parameter optimization using experimental design principles, which revealed the key influence of fragmentor voltage for this application. Datasets from alternating in-source fragmentation high resolution mass spectrometry (HRMS) were evaluated using open-source data processing tools combined with public reference mass spectral databases. The significant influence of the selected fragmentor voltages on the abundance of the primary analyte ion of interest and the extent of in-source fragmentation allowed an optimum selection of qualifier fragments for the different metabolites. The new acquisition and evaluation workflow was implemented for the non-targeted analysis of yeast extract samples whereby more than 130 metabolites were putatively annotated with more than 40% considered to be of high confidence. The presented workflow contains a fully elaborated acquisition and evaluation methodology using alternating in-source fragmentor voltages suitable for peak annotation and metabolite identity confirmation for non-targeted metabolomics applications performed on a single-stage HRMS platform.

Immobilized bacterial biosensor for rapid and effective monitoring of acute toxicity in water.

The use of biosensors by using microorganisms such as bacteria have short life cycles and provide other advantages. One colorimetric biosensor technique that has been developed is the use of a biosensor utilizing the incorporation of Prussian blue formation reactions mediated by E. coli bioreactors with ferricyanide. Immobilization is a method that allows the bacteria can be used for long-term without reducing its ability as bioreceptor. This study aimed to develop a novel and rapid immobilized bacterial biosensor for the detection of toxic compound in water and to evaluate their analytical performances. Immobilization of E. coli performed by trapping method using alginate material support. The bacterial suspension was mixed with sodium alginate (1:1 v/v), and the mixture was continuously dropped in CaCl2 solution to be a form of beads. The beads were used as bioreceptor to detect toxicants regarding cadmium, arsenic, mercury, chromium and lead solutions with Prussian blue as a colorimetric indicator. The linearity and sensitivity of detection of beads to the toxicants were tested, the stability of repeated use and storage were evaluated as well. The results showed that E. coli could be immobilized using alginate with response value was correlated with toxic concentration. The developed biosensor was more stable when used repeatedly and could be stored in a long time. The immobilization of E. coli in calcium alginate bead was successfully performed as a biosensor system for monitoring acute toxicity in water.