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A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectr...

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Detalles Bibliográficos
Autores principales: Sasidharan, Kalesh, Soga, Tomoyoshi, Tomita, Masaru, Murray, Douglas B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3430680/
https://www.ncbi.nlm.nih.gov/pubmed/22952947
http://dx.doi.org/10.1371/journal.pone.0044283
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author Sasidharan, Kalesh
Soga, Tomoyoshi
Tomita, Masaru
Murray, Douglas B.
author_facet Sasidharan, Kalesh
Soga, Tomoyoshi
Tomita, Masaru
Murray, Douglas B.
author_sort Sasidharan, Kalesh
collection PubMed
description There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.
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spelling pubmed-34306802012-09-05 A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses Sasidharan, Kalesh Soga, Tomoyoshi Tomita, Masaru Murray, Douglas B. PLoS One Research Article There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously. Public Library of Science 2012-08-29 /pmc/articles/PMC3430680/ /pubmed/22952947 http://dx.doi.org/10.1371/journal.pone.0044283 Text en © 2012 Sasidharan et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Sasidharan, Kalesh
Soga, Tomoyoshi
Tomita, Masaru
Murray, Douglas B.
A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses
title A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses
title_full A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses
title_fullStr A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses
title_full_unstemmed A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses
title_short A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses
title_sort yeast metabolite extraction protocol optimised for time-series analyses
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3430680/
https://www.ncbi.nlm.nih.gov/pubmed/22952947
http://dx.doi.org/10.1371/journal.pone.0044283
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