Gas Chromatography - Mass Spectrometry and Metabolomic Analysis
Algae were grown in three biological replicates at 4°C, 10°C and 15°C and sampled at a steady-state temperature or after 6h exposure to 24°C. Cells were harvested by centrifugation (6,000g, 5 min), washed once with fresh medium, flash frozen in liquid N2 and stored at -80°C. Metabolite extractions, chromatography and quality processing were done at the West Coast Metabolomics Center (UC Davis, CA, USA), following a previously established protocol optimized for retention and separation of primary metabolite classes (amino acids, carbohydrates, sugar acids, sterols, aromatics, nucleosides, amines and miscellaneous compounds (Fiehn et al. 2008). Mass spectra were processed using BinBase, and analysed as described in (Fiehn, Wohlgemuth & Scholz 2005). Metabolites were identified based on their mass spectral characteristics and GC retention times by comparison with compounds in a plant and algae reference library (West Coast Metabolomics Center). Peak heights for the quantification ion at the specific retention index corresponding to each metabolite were normalized by the sum of peak heights in the sample. Normalized data were processed by cube root transformation followed by range scaling. All statistical analyses were performed by the Metaboanalyst 4.0 software suite (Chong et al.2018) and included principal component analysis (PCA), analysis of variance (ANOVA), heatmap and clustering analysis using Ward’s linkage for clustering and Pearson’s correlation as a measure of dissimilarity.