Sensitive quantitative analysis of phosphorylated primary metabolites using selective metal oxide enrichment and GC- and IC- MS/MS

Si-Hung, Le; Troyer, Christina; Causon, Tim J.

doi:10.1016/j.talanta.2019.120147

Cited by 19 publications

(15 citation statements)

References 42 publications

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“…Phaff 1956 (Komagataella phaffii Kurtzman) 27 Literature search confirmed our findings and additionally expanded the reported compounds [30][31][32][33][34][35][36][37][38][39] in our library which can be found in the Supplementary two participating laboratories or others [30][31][32][33][34][35][36][37][38][39] ). The remaining 25 metabolites were reported elsewhere [30][31][32][33]36,37 ( Supplementary Table S1, column, FinalMetaboliteList, "Additional literature hits"). The human metabolome database (HMDB) 43,44 was interrogated regarding overlaps with the yeast metabolome data base which is based on the Baker's/Brewer's yeast Saccharomyces cerevisiae 45 crosschecking our library for plausibility.…”

Section: Metabolites and Lipids Present In The Yeast Materialssupporting

confidence: 80%

“…Over the recent past, yeast-based standards have seen a slow but increasing acceptance in the metabolomics community. Several laboratories resorted to Pichia pastoris ethanolic yeast extracts [30][31][32][33][34][35][36][37][38][39] primarily for 13 C internal standardization. Only a few studies were related to instrument performance tests or method development in accordance to the here proposed application 33,[38][39][40] .…”

Section: Resultsmentioning

confidence: 99%

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Benchmarking non-targeted metabolomics using yeast derived libraries

Rampler

Hermann

Grabmann³

et al. 2020

Preprint

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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.

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Section: Metabolites and Lipids Present In The Yeast Materialssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Benchmarking non-targeted metabolomics using yeast derived libraries

Rampler

Hermann

Grabmann³

et al. 2020

Preprint

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“…GC–MS/MS followed by just in time two-step derivatization was ideally suited to absolutely quantify intermediates of the glycolysis and pentose phosphate pathway due to the unrivalled selectivity of the chromatographic separation allowing to separate sugar phosphate isomers. Supplementary Table S1–S2 detail the metabolite targets investigated by HILIC-HRMS and GC–MS/MS, respectively [ 24 , 33 , 34 ]. The technical uncertainty of the methods was on average < 10% (as exemplified with the uncertainty calculation for the amino acid threonine in the supplement); thus, the methods were fit for purpose.…”

Section: Resultsmentioning

confidence: 99%

“…Small molecules' targeted data evaluation was carried out with the open-source software Skyline 20.1.0.76 [22]. GC-MS/MS measurement was performed to determine other intracellular metabolites that were not covered by HILIC-HRMS, adapting the procedure used by Mairinger et al and Si-Hung et al [23,24]. In short, just in time online two-step derivatization was performed automatically using a Gerstel MPS2 dual-rail sample preparation robot (Gerstel GmbH, Muehlheim, Germany).…”

Section: Hilic-hrms and Gc-ms/ms Measurementsmentioning

confidence: 99%

“…Being derived from the same microorganism and produced by an equivalent fermentation using 13 C-glucose as carbon source, the material offered the ideal match regarding both the composition and concentration ranges. The applied isotope dilution strategy (external calibration with 13 C internal standards) enabled accurate quantification despite multiple sample preparation steps, as shown in different targeted metabolomics studies [23,24,26]. Volume losses during sample preparation or instrumental drifts could be compensated [18].…”

Section: Characteristics Of Reference Materialsmentioning

confidence: 99%

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Yeast-based reference materials for quantitative metabolomics

et al. 2021

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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.

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A simple ion chromatography‐mass spectrometry method for amino acid analysis in beer

Schmitt

Fillsack

Seubert

2023

J of Separation Science

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The amino acid footprint of different beer samples was analyzed using ion chromatography coupled with electrospray ionization mass spectrometry. A tailor-made polymer-based cation-exchange resin was operated with a mass spectrometry-compatible eluent under isocratic conditions on a standard highperformance liquid chromatography system coupled to a single quadrupole mass spectrometer using formic acid as a volatile eluent ion source. The partially separated peaks of the isomeric pair isoleucine/leucine were processed according to their area response ratio using vertical peak splitting or Gaussian fit. Additionally, the chromatographic resolution of the isomers was optimized with an adjusted, solely aqueous mobile phase from 0.85 to 2.92. Ion suppression in the electrospray ion source was investigated for the derivatization-free method and found to be insignificant (recovery value 100 ± 15%) for 15 out of the 20 analytes.Quantitative results for various beer and mixed-beer beverages were found to be in high agreement with existing methods. Simultaneous photometric detection demonstrated the method's ability to successfully remove most of the interfering matrix compounds.

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Sensitive quantitative analysis of phosphorylated primary metabolites using selective metal oxide enrichment and GC- and IC- MS/MS

Cited by 19 publications

References 42 publications

Benchmarking non-targeted metabolomics using yeast derived libraries

Benchmarking non-targeted metabolomics using yeast derived libraries

Yeast-based reference materials for quantitative metabolomics

A simple ion chromatography‐mass spectrometry method for amino acid analysis in beer

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