Gas Chromatography - Mass Spectrometry and Metabolomic Analysis
For determination of the primary metabolome, algae were grown in three
biological replicate cultures as described above and sampled at a
steady-state temperature or after 6h exposure to heat stress. Algal
cells were harvested by centrifugation (6,000g, 5 min), washed once with
fresh medium, flash frozen in liquid nitrogen 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 (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, UC Davis, CA, USA). 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 (van den Berg, Hoefsloot, Westerhuis, Smilde & van der
Werf 2006). 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.