Dataset: Phytoplankton exometabolite concentrations from laboratory cultures of Crocosphaera watsonii, Micromonas commoda, Prochlorococcus marinus, Synechococcus, Gephyrocapsa huxleyi, and Thalassiosira pseudonana collected September 2022- December 2022

Standards, filtrate samples, and media blanks were derivatized with benzoyl chloride (BC) (Widner et al., 2021). The derivatization and sample preparation protocols together with pictures and video clips are available online (https://www.protocols.io/workspaces/kujawinski-lab). Briefly, each 5-milliliter (mL) sample was basified with 150 microliters (µL) of 8 M NaOH, derivatized with 1 mL BC working solution for 5 minutes, and then acidified with 75 µL phosphoric acid. The BC working solution was prepared by mixing 95 mL acetone (Optima, ACROS Organics) with 5 mL BC (99%, ACROS Organics) and was used within 36 hours after preparation. Two sets of stable isotopically labeled internal standards (SIL-IS) were prepared by derivatizing standard mixes of metabolites in seawater (aged, 0.1-µm filtered seawater collected from offshore Caribbean Sea) using BC working reagent prepared with ¹³C₆-BC (99% ¹³C, Sigma-Aldrich) or D₅-BC (99% D, Cambridge Isotope Laboratories, Inc.), respectively. SIL-IS were derivatized following the same protocol as the samples.

Each derivatized standard, filtrate, or media blank was spiked with 498 pg addition of ¹³C₆-labeled SIL-IS and 9.96 nanograms (ng) addition of D₅-labeled SIL-IS and was dried using a vacufuge (Eppendorf) until ≥ 95% (by weight) of acetone was removed. Upon acetone removal, the liquid was transferred onto a preconditioned (6 mL of methanol followed by 24 mL of 0.01 M HCl) Bond Elut PPL cartridge (1g/6 mL, Agilent) and loaded by gravity. The samples were eluted with 6 mL of methanol by vacuum followed by evaporation to near dryness in a vacufuge. Samples were reconstituted with 100 µL 5% acetonitrile in Milli-Q water, vortexed thoroughly, and then centrifuged at 12000 g, 22 degrees Celsius (°C) for 5 minutes. The supernatant was transferred to LC vials with glass inserts, which were previously spiked with 5 µL of acetonitrile. The samples were stored at 4°C until LC-MS analyses.

Targeted metabolomics analyses were performed using an ultrahigh performance liquid chromatography system (Vanquish UHPLC, Thermo Scientific) coupled to a heated electrospray ionization source (H-ESI) and an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific). A Waters Acquity HSS T3 column (2.1 mm × 100 mm, 1.8 µm), equipped with an Acquity HSS T3 VanGuard Pre-column, was used for chromatographic separation at 40°C. The column was eluted at 0.5 mL per minute with a combination of solvents: A) 0.1% formic acid in water and B) 0.1% formic acid in acetonitrile. The chromatographic gradient was as follows: 1% B (0-0.5 min), 10% B (0.5-2.0 min), 10% B (2.0-5.0 min), 25% B (5.0-7.0 min), 25% B (7.0-9.0 min), 50% B (9.0-12.5 min), 95% B (12.5-13.0 min), 95% B (13.0-14.5 min), 1% B (14.5-14.6 min), and 1% B (14.6-16.0 min). The autosampler was set at 4°C. Individual autosampler injections (5 µL each) were used for negative and positive ion mode analyses. The first 0.8 minutes of flow was diverted to waste after passing through the column. Other instrument parameters were: ESI voltages = 3600 V (positive) and 2600 V (negative); source gases = 55 (sheath), 20 (auxillary), and 1 (sweep); capillary temperature = 350°C; vaporizer temperature = 400°C. MS data from 170-1000 m/z were collected at resolution 60,000 FWHM (at m/z 200), automatic gain control (AGC) at 4e5, and max injection time 50 msec. MS/MS data were triggered using a targeted mass list with m/z and retention time window for each derivatized analyte, and collected at resolution 7,500 FWHM, AGC 5e4, and max injection time 22 milliseconds using higher energy collisional dissociation (HCD) with 35% collision energy and intensity threshold 2e4. Parent ions were isolated within the quadrupole at a width of 1 m/z.