NMR analysis of budding yeast metabolomics: a rapid method for sample preparation

Airoldi, Cristina; Tripodi, Farida; Guzzi, Cinzia; Nicastro, Raffaele; Coccetti, Paola

doi:10.1039/c4mb00452c

Cited by 23 publications

(23 citation statements)

References 21 publications

(21 reference statements)

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“…22 Dried samples were stored at −80°C until the NMR analysis. Then, cells were resuspended in 3 mL of 75% ethanol and 2 mL of 450 to 600 μm diameter glass beads.…”

Section: Polar Metabolite Extractionmentioning

confidence: 99%

¹H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

Rona

Almeida

Santos

et al. 2018

J of Cellular Biochemistry

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NSD3s, the proline‐tryptophan‐tryptophan‐proline (PWWP) domain‐containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using 1H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein‐dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.

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“…22 Dried samples were stored at −80°C until the NMR analysis. Then, cells were resuspended in 3 mL of 75% ethanol and 2 mL of 450 to 600 μm diameter glass beads.…”

Section: Polar Metabolite Extractionmentioning

confidence: 99%

¹H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

Rona

Almeida

Santos

et al. 2018

J of Cellular Biochemistry

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“…Boiling ethanol, first introduced as an approach to extract metabolites from yeast, has become one of the most frequently used methods for metabolomics studies in other biological systems (Airoldi et al 2015; Dunn and Winder 2011; Gonzalez et al 1997). While several researchers have used this type of extraction with different modifications, according to particular needs (Buescher et al 2010; Canelas et al 2009; Faijes et al 2007; Paglia et al 2012; Sellick et al 2010), there is little information regarding the stability of critical molecules, including nucleotides, under such harsh extraction conditions.…”

Section: Introductionmentioning

confidence: 99%

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix

et al. 2016

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IntroductionBoiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.ObjectivesTo study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.MethodsWe present a double-labelling LC–MS approach with a 13C-labeled yeast cellular extract as complex surrogate matrix, and 13C15N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.ResultsWhile nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.ConclusionsExtraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.Graphical abstract Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-016-1140-4) contains supplementary material, which is available to authorized users.

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“…Problems of metabolite loss by cell leakage and extracellular metabolite contamination were observed in some cases (Airoldi et al 2015). Ideally, all of the intracellular metabolites are completely extracted in a reproducible manner.…”

Section: Sample Preparation For Yeastmentioning

confidence: 99%

“…Ideally, all of the intracellular metabolites are completely extracted in a reproducible manner. Some researchers omit the quenching procedure altogether or use fast filtration instead of quenching (Airoldi et al 2015). The use of cold methanol quenching has been used to arrest cellular metabolism in S. cerevisiae and is still commonly used as a quenching method for yeast.…”

Section: Sample Preparation For Yeastmentioning

confidence: 99%

“…The extractions are routinely performed at low temperature in order to preserve the metabolome. Washing the cells was found to be critical for obtaining reproducible and reliable data (Airoldi et al 2015). There have been only a handful of studies that have assessed the metabolite loss or the effect of washing after cold methanol quenching in S. cerevisiae.…”

Section: Sample Preparation For Yeastmentioning

confidence: 99%

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Metabolomics approaches for resolving and harnessing chemical diversity in grapes, yeast and wine

Lloyd

Johnson

Herderich

2015

Australian Journal of Grape and Wine Research

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Metabolomics is the scientific study of the unique chemical fingerprints that result from specific biochemical processes. It enables the comprehensive detection and quantification of small molecules that are intermediates or end‐products of metabolism in a biological sample, such as a grape or yeast. The metabolomics approach also provides for a holistic characterisation of complex chemical transformations and oxidation reactions during winemaking, ageing and transport. The development of metabolomics has made major advances in the last two decades. Improved chromatographic techniques for separation of polar metabolites and enhanced sensitivity and selectivity of nuclear mass resonance and mass spectrometric instrumentation now enable detection and quantification of hundreds of metabolites in a single analysis. New bioinformatics tools and statistics capabilities are available which are key to efficiently analysing and annotating comprehensive datasets. This review provides an overview of analytical methods for untargeted profiling, biomarker discovery and targeted analysis of non‐volatile and volatile metabolites. It summarises bioinformatics tools for data processing, data mining and metabolite identification and explores metabolomics approaches which have been successfully employed to comprehensively analyse, annotate and understand the broad chemical diversity in grapes, yeast and wine. It also describes opportunities for the application of metabolomics in grape and wine science and provides an outlook about further development needs and emerging trends.

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NMR analysis of budding yeast metabolomics: a rapid method for sample preparation

Cited by 23 publications

References 21 publications

¹H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

¹H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix

Metabolomics approaches for resolving and harnessing chemical diversity in grapes, yeast and wine

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NMR analysis of budding yeast metabolomics: a rapid method for sample preparation

Cited by 23 publications

References 21 publications

1H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

1H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix

Metabolomics approaches for resolving and harnessing chemical diversity in grapes, yeast and wine

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¹H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

¹H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae