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.
Extraction protocol flow diagram.
Table 1.
Metabolites (attomol/cell) extracted from samples taken during respiratory oscillation (minimum dissolved oxygen; figure 4), detected by CE-MS and the methods used to extract them. nd – not detected.
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.
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.