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GCMS agar samples from plates with S. coelicolor sampled every 3rd day for 27 days

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posted on 2024-04-25, 11:31 authored by Linda SandbladLinda Sandblad, Niklas SöderholmNiklas Söderholm

For the nutrient analysis of agar, six replicates of agar plugs were collected from the edge of the dish and 0.5 cm away from the S. coelicolor colonies on 0.5xTSA medium. This sampling was performed on six different ⌀ 4 cm plates. Three control samples were taken at every time point from three sterile 0.5xTSA dishes. Sample preparation was performed according to A et al. (A et al., 2005). The samples were stored at -80 °C until analysis. Small aliquots of the remaining supernatants were pooled and used to create quality control (QC) samples. The samples were analyzed in batches according to a randomized run order on GC-MS. Derivatization and GCMS analysis were performed as described previously A et al. (A et al., 2005). Non-processed MS-files from the metabolic analysis were exported from the ChromaTOF software in NetCDF format to MATLAB-R2020a (Mathworks, Natick, MA, USA), where all data pre-treatment procedures (base-line correction, chromatogram alignment, data compression and Multivariate Curve Resolution) were performed. The extracted mass spectra were identified by comparisons of their retention index and mass spectra with libraries of retention time indices and mass spectra (Schauer et al., 2005). Mass spectra and retention index comparison was performed using NIST MS 2.2 software. Annotation of mass spectra was based on reverse and forward searches in the library. Masses and ratio between masses indicative of a derivatized metabolite were especially notified. The mass spectrum with the highest probability indicative of a metabolite and the retention index between the sample and library for the suggested metabolite was ± 5 the deconvoluted “peak” was annotated as an identification of a metabolite.

Funding

Kempestiftelserna

Sven och Lilly Lawskis fond

History

Publisher

Umeå Universitet

SciLifeLab acknowledgement

  • Swedish Metabolomics Centre unit

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