Abstract
A metabolite profiling technique for Chlamydomonas reinhardtii cells for multiparallel analysis of low-molecular weight polar compounds was developed. The experimental protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity, and process large sample quantities. As a result of the rapid sampling, extraction, and analysis by gas chromatography coupled to time-of-flight mass spectrometry, more than 800 analytes from a single sample could be measured, of which more than 100 could be identified. Analyte responses could be determined mostly with ses less than 10%. Wild-type cells of C. reinhardtii strain CC-125 subjected to nitrogen-, phosphorus-, sulfur-, or iron-depleted growth conditions develop highly distinctive metabolite profiles. Individual metabolites undergo marked changes in their steady-state levels. Compared to control conditions, sulfur-depleted cells accumulated 4-hydroxyproline more than 50-fold, whereas the amount of 2-ketovaline was reduced to 2% of control levels. The contribution of each compound to the differences observed in the metabolic phenotypes is summarized in a quantitatively rigorous way by principal component analysis, which clearly discriminates the cells from different growth regimes and indicates that phosphorus-depleted conditions induce a deficiency syndrome quite different from the response to nitrogen, sulfur, or iron starvation.
Highlights
A metabolite profiling technique for Chlamydomonas reinhardtii cells for multiparallel analysis of low-molecular weight polar compounds was developed
Because of unsurpassed chromatographic separation power, sensitivity, and reproducibility, we have chosen gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) to establish a method for analysis of metabolite levels of the unicellular green alga C. reinhardtii
To obtain metabolomic data that correctly measure amount of substance and reflect the levels of intracellular metabolites, we followed a multistep procedure that conceptually can be divided into cell harvest and extraction, sample preparation for the acquisition of
Summary
To obtain metabolomic data that correctly measure amount of substance and reflect the levels of intracellular metabolites, we followed a multistep procedure that conceptually can be divided into cell harvest and extraction, sample preparation for the acquisition of. Plant Physiology, December 2005, Vol 139, pp. 1995–2005, www.plantphysiol.org Ó 2005 American Society of Plant Biologists Downloaded from on October 5, 2020 - Published by www.plantphysiol.org
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