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PLoS By Category | Recent
PLoS Articles
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Biochemistry - Microbiology - Physiology
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A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses
Published:
Wednesday, August 29, 2012
Author:
Kalesh Sasidharan et al.
by Kalesh Sasidharan, Tomoyoshi Soga, Masaru Tomita, Douglas B. Murray
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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