Untargeted metabolomic analysis of harvested samples was done by Michigan Regional Comprehensive Metabolomics Resource Core (MRC2) (Ann Arbor, MI, USA). Metabolite extraction was done in Methanol: Acetonitrile: Acetone (1:1:1) solution with internal standards.
Samples were reconstituted in solvent containing Methanol and H2O (2:98). 100 µl sample was added to 400 µl extraction solvent and reconstituted in 100 µl reconstitution solvent. For untargeted metabolomics, samples were analyzed on 1290 Liquid chromatography, coupled with 6530 qTOF mass spectroscopy (Agilent Technologies, Santa Clara, CA, USA). Same chromatography was used for positive and negative modes. Raw data was processed using MassHunter Qual and ProFinder software (Agilent), and analyzed with MassProfiler Pro package using recursive analysis workflow (Agilent). Data normalization was performed using custom R-scripts (internal to Michigan Regional Comprehensive Metabolomics Resource Core (MRC2) (Ann Arbor, MI, USA). 243 annotated compounds were identified in our sample by comparing the observed mass and retention time to the in-house library. Remaining unannotated compounds were listed with their observed mass and retention times.
Samples were reconstituted in solvent containing Methanol and H2O (2:98). 100 µl sample was added to 400 µl extraction solvent and reconstituted in 100 µl reconstitution solvent. For untargeted metabolomics, samples were analyzed on 1290 Liquid chromatography, coupled with 6530 qTOF mass spectroscopy (Agilent Technologies, Santa Clara, CA, USA). Same chromatography was used for positive and negative modes. Raw data was processed using MassHunter Qual and ProFinder software (Agilent), and analyzed with MassProfiler Pro package using recursive analysis workflow (Agilent). Data normalization was performed using custom R-scripts (internal to Michigan Regional Comprehensive Metabolomics Resource Core (MRC2) (Ann Arbor, MI, USA). 243 annotated compounds were identified in our sample by comparing the observed mass and retention time to the in-house library. Remaining unannotated compounds were listed with their observed mass and retention times.