1P-38 PDF
LC-MSを用いた酵母中心代謝物質の絶対定量
An absolute quantification method was developed for a precise analysis of central metabolic intermediates in Saccharomyces cerevisiae. A mixture of 13C labeled metabolites was prepared from S. cerevisiae cells grown in [U-13C] glucose. To prepare stable isotope labeled central metabolic intermediates, an extract of U-13C labeled intermediates was obtained from S. cerevisiae (S288C strain) cells cultured in a medium containing [U-13C] glucose. Concentrations of each intermediate in the 13C labeled extract were determined by an LC-MS/MS analysis of the extract mixed with a solution of non-labeled standard compounds. Using the U-13C labeled extract as an internal standard mixture, levels of 13 intermediates in S. cerevisiae cells cultured in a non-labeled medium were successfully determined using LC-MS/MS. Using the absolute concentration data, levels of Gibb’s free energy (ΔG) were calculated for ribose-5-phosphate isomerase and ribose-5-phosphate 3-epimerase. Since levels of ΔG in BY4742, pfk1Δ and zwf1Δ were closed to 0, it was suggested that these reactions were at near equilibrium and unlikely to be rate-limiting steps.