Dataset: Shotgun Proteomics of Pseudonitzschia multiseries Multi stress incubations.

Bottles were removed from the incubators, and the sampling ports carefully disconnected within the laminar flow hood. The cells were then re-suspended by gently inverting each bottle. Samples for pH were taken after the bottles were returned to the incubator. Sampling for all experimental parameters in each bottle was carried out on day 0, 4 and 9 (all treatments), and then days 11 and 14 for treatments B and D and on day 15 for A and day 17 for A and C. The following protocols were employed at each sampling point. For cell counts 1 ml samples were fixed with 50% glutaraldehyde to a final concentration of 0.5% and stored at 4 °C. Cells were counted with an Olympus CKX 41 inverted microscope using a 0.1 ml nannoplankton chamber (PhycoTech). Protocols for in vivo and in vitro chlorophyll analysis, active fluorescence, and the calculation of chlorophyll-based cell growth rates followed those in refs 56, 57. Cellular particulate C and N were analysed in a Thermo Flash 2000 CHN Elemental Analyser. Particulate P and biogenic Si were analysed following procedures in ref. 60 and ref. 61, respectively, and converted to cellular elemental composition based on cell counts. The low cell abundances in treatment C resulted in some assays being close to the limits of detection and in some cases being lower than the blanks. No subsamples were taken for dissolved iron analysis during the experiment, but confirmation of no trace-metal contamination of the treatments was obtained indirectly by monitoring several physiological metrics—such as C/chlorophyll, growth rate or cellular silica (see Supplementary Table 4)—that are sensitive to iron supply.

Data-dependent tandem mass spectrometry was carried out on a Thermo Scientific Q-Exactive tandem mass spectrometer following protocols detailed in (Poulson-Ellestad, K. L. et al. Metabolomics and proteomics reveal impacts of chemically mediated competition on plankton. Proc. Natl Acad. Sci. USA 111, 9009–9014 (2014).

Cells were pelleted (10,000 × g; 10 min) on ice and lysed using a titanium microtip sonicating probe. Each sample received 10 sonication events (10–15 s each) in 0.2% sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl) propane-1-sulfonate (PPS silent surfactant; Agilent Technologies)  in 50 mM ammonium bicarbonate. The details of the digestion were per the manufacturer’s guidelines. Disulfide bonds were reduced with DTT and alkylated with iodoacetamide. Each sample received trypsin at an enzyme-to-protein ratio of 1:50, were vortexed, and were incubated on a Thermomixer for 4 h at 37 °C. Peptide concentrations were measured for each sample using a Thermo Scientific NanoDrop 2000/2000c spectrophotometer. The peptide bond absorbance was monitored at 205 nm, and samples were diluted to yield a final concentration of 100 μg protein·mL−1 . Mass Spectrometry-Based Proteomics. Samples were separated and introduced into the Thermo Scientific Q-Exactive tandem mass spectrometer by reversed-phase chromatography using a 30-cm-long, 75-μm-i.d. fused silica capillary column packed with C18 silica particles (Magic C18AQ, 100 Å, 5 μ; Michrom, Bioresources) fitted with a 2-cm-long, 100-μm-i.d. precolumn (Magic C18AQ, 200 Å, 5 μ; Michrom) . Peptides were eluted using an acidified [formic acid, 0.1% (vol/vol)] water/acetonitrile gradient (2–35% acetonitrile over 90 min). Mass spectrometry was performed on a Thermo Fisher QExactive (QE). Based on peptide concentrations, a total of 1 μg of peptide digest in 10 μL of 2% acetonitrile, 0.1% formic acid was sampled per LC/MS analysis.