Flowchart of approaches employed to putatively annotate PMSs secreted from oral bacteria. Flowchart of approaches employed to putatively annotate PMSs.

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Flowchart of approaches employed to putatively annotate PMSs secreted from oral bacteria. Flowchart of approaches employed to putatively annotate PMSs secreted from oral bacteria. (Step 1) A highly diverse in vitro biofilm community and single isolates of bacteria were grown in 1-ml cultures in a 24-well format. For the experimental setup, see Fig. S1. To identify parent masses and their corresponding ion fragment profiles in each sample, growth extracts were analyzed with an UltiMate 3000 UHPLC system and a Maxis qTOF mass spectrometer equipped with an electrospray ionization (ESI) source. (Steps 2A and B) Parent masses obtained from replicate samples were sorted into bucket tables and compared between time points (step 2A) and between bacterial isolates (step 2B) by using Venn diagrams and cluster analyses. (Steps 3A and B) The Global Natural Products Social Networks infrastructure (22) was employed to putatively annotate MS/MS spectra by spectral alignments of query spectra with ~20,000 benchmarked MS/MS spectra in the GNPS library (step 3A). GNPS networks revealed associations between query spectra and benchmark spectra, which contributed to level 2 annotations of ~50 PSMs (step 3B). (Steps 4A and B) The DEREPLICATOR tool (23) was used to annotate MS/MS spectra and predict the probability of each annotation by calculating false discovery rate (FDR) scores (step 4A). These annotations were based on structural homologies with PMS in databases such as PubMed and correspond to level 2 annotation standards (step 4B). Anna Edlund et al. mSystems 2017; doi:10.1128/mSystems.00058-17