GNPS - StM16 standards mixture spiked into Pseudomonas aeruginosa extract

Preparation of PA14 extract spiked with StM16-Mix at different concentrations.
A mixture of 16 compounds - 2-methoxybenzoic acid, biochanin A, trans-ferulic acid, 3-indoleacetonitrile, indole-3-carboxaldehyde, kinetin, p-coumaric acid, L-(+)-alpha-phenylglycine, phloridzin dihydrate, rutin trihydrate, indole-3-acetyl-L-valine, quercetin, naproxen, nortriptyline, trimethoprim, and novobiocin - was prepared at a concentration of 120 uM per compound in a 1:1 mixture of acetonitrile and water.
The StM16-Mix was then spiked into a Pseudomonas aeruginosa PA14 extract, prepared according to Franke et al. (mSystems, 2021), to achieve final concentrations of 10, 8, and 4 uM.

LC-MS analysis. For each concentration, 1 uL of the StM16-spiked PA14 extract was analyzed by reversed phase ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with n= 3 technical replicates. The samples were analyzed by positive mode electrospray ionization quadrupole time-of-flight mass spectrometry on a maXis HD QTOF (Bruker, Bremen, Germany) in full scan mode (50 to 1500 Da). Accurate masses were obtained by internal calibration using an ion cluster of sodium formate and lock mass calibration. The StM16-Mix was also analyzed using data-dependent MS/MS by collision-induced dissociation of the five most abundant ions in each scan, making use of the Bruker smart exclusion algorithm.