Metabolite target analysis of isoprenoid pathway in Saccharomyces cerevisiae in response to genetic modification by GC-SIM-MS coupled with chemometrics

Abstract

A highly selective, sensitive and nonradioactive analytical method for identification and quantification of intracellular metabolites involved in isoprenoid pathway has been developed by means of gas chromatography-selected ion-monitoring mass spectrometry (GC-SIM-MS). These metabolites are classified into two groups: sterols (squalene, ergosterol, lanosterol) and phosphorylated compounds (geranyl diphosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate) based on their physicochemical properties. To quantify both groups in a single analytical run, GPP, FPP and GGPP were cleaved to the parent alcohols, geraniol, farnesol, geranylgeraniol by pyrophosphatase followed by alkaline phosphatase before extraction, separation and detection. This study evaluated several extraction procedures and determined the effects of the type of extraction solvent, times used for extraction. Under optimized GC/EI-MS conditions, six compounds were separated with high efficiency in the selected-ion monitoring (SIM) mode. Linearity of the method was good with correlation coefficients (r 2) in the range of 0.9953–0.9999 and detection limits were 1.53–151.88 ng/ml. The intra-day and inter-day precision of the method, as RSD, were less than 5.31 and 6.04%, respectively. The accuracy of six compounds varied between 87.7 and 110.8%. This assay was successfully applied to the determination of six major metabolites in the pathway for isoprenoid biosynthesis in S. cerevisiae and is sensitive to detect changes following genetic modification. By isolating statistically significant differences among metabolite levels from four biological conditions, we observed discriminatory metabolic features that hinted that the role of erg9 and coq1gene was involved in isoprenoid pathway. Integrating this analytical approach with statistical strategies, we can determine the influence of erg9 and coq1gene on isoprenoid levels of S. cerevisiae, thus leading to improved understanding of the pathway in a multitude of biological systems.