2.15. Metabolomics and data analysis

Spectral data were extracted and aligned based on mass and retention time using MassHunter Profinder software B.08.00 (Agilent). The parameters for the molecular feature extraction included: a peak height of ≥1500 ion counts and possible ions with [M + H]⁺. The alignment also involved isotope grouping restrictions including: a peak spacing tolerance of 0.0020 m/z and 10.0 ppm and a maximum charge state of 1. It was also required that there were at least two or more ions for a single molecular feature. In addition, for binning and alignment purposes, a tolerance of 0.3 min for a retention time window was set along with a mass window of 20 ppm. Some of the post processing filters included: an absolute height filter of ≥3000 ion counts and the requirement for the molecular feature to be present in at least two out of three replicates in one experimental group.

The resulting aligned features were analyzed in MetaboAnalyst 4.0 (www.metaboanalyst.ca) and SIMCA-P + 14.0 (Umetrics, Umea, Sweden) for statistically significant differentiations between sampling conditions. The resulting m/z's were searched in METLIN database to determine possible candidate metabolites based on accurate mass and adduct.