UC Davis

The green eukaryote alga Chlamydomonas reinhardtii is a unicellular model to study control of metabolism in a photosynthetic organism. We here present method improvements for metabolite profiling based on GC-TOF mass spectrometry focusing on three parameters: quenching and cell disruption, extract solvent composition and metabolite annotation. These improvements facilitate using smaller cell numbers and hence, smaller culture volumes which enable faster and more precise sampling techniques that eventually lead to a higher number of samples that can be processed, e.g. for time course experiments. Quenching of metabolism was achieved by mixing 1 ml of culture to 1 ml of -70 degrees C cold 70% methanol. After centrifugation, cells were lyophilized and disrupted by milling using 2-6E6 lyophilized cells, around 500-fold less than previously reported. Glass beads were compared to metal balls for milling, and five different extraction solvents were tested. Additionally, all peaks were annotated in an automated way using the GC-TOF database BinBase instead of manual investigation of a single reference chromatogram. Median precision of analysis was used to decide for the eventual procedure which was applied to a proof-of-principle study of time dependent changes of metabolism under standard conditions.