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. 2012;7(8):e44283.
doi: 10.1371/journal.pone.0044283. Epub 2012 Aug 29.

A yeast metabolite extraction protocol optimised for time-series analyses

Kalesh Sasidharan  1 , Tomoyoshi Soga, Masaru Tomita, Douglas B Murray
Affiliations

A yeast metabolite extraction protocol optimised for time-series analyses

Kalesh Sasidharan et al. PLoS One. 2012.

Abstract

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The general reaction scheme for N-ethylmaleimide on biological thiols.
The product formed is an N-ethylsuccinimido conjugate and the monoisotopic mass of the reactant is increased by 125.048. For example the glutathione (m/z 308.0911) conjugate is N-ethylsuccinimido-S-glutathione (m/z 433.1391).
Figure 2
Figure 2. Extraction protocol flow diagram.
Figure 3
Figure 3. Electrophoretograms of glutathione (GSH) and homocysteine (Hcys) and their NEM derivatives (ESG and ESHcys) in representative samples with (red line) and without (green line) the addition of 2 mM NEM.
Figure 4
Figure 4. A heatmap of the calibrated intracellular metabolite time-series during the respiratory oscillation (A) and time-series for ATP (B), glutathione (C), and aspartate (D) in S. cerevisiae.
Cationic and anionic data are shown in red and blue (lines or shaded areas), respectively. The corresponding oscillation in dissolved oxygen (DO) is represented by a grey line.
See this image and copyright information in PMC

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