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.