Experimental design

Synechococcus cultures (WH8102) were grown in polycarbonate bottles with a continuous chemostat method used previously (Garcia et al. 2016) in artificial seawater. We used two concentration ratios of macronutrients (NO3-:PO43- = 1.7 and 80) and 3 levels of temperature (20, 24 and 28°C) with a slow dilution rate (0.18 d-1) to ensure treatment-wise culture stability. Target concentrations of NO3- and PO43- in chemostats were 45 µM and 0.56 µM, respectively in the N:P=80 treatment, and 15.9 µM and 9.2 µM, respectively in the N:P=1.7 treatment. White light was supplied at 125 µmol quanta m-2 s-1 on a 12h:12h light:dark cycle. Chemostat equilibria were monitored by measuring culture cell density and forward scatter (FSCH) with a Novocyte flow cytometer 1000 (Acea Biosciences, Inc, San Diego, CA). Cells for proteome analysis were collected after an acclimation period on days 38, 43, 47, 50 and 57 with a 47 mm polycarbonate filter (0.2 µm pore size) 7-8 hours into the light period, pelleted by centrifugation (21,130 g for 3 minutes), flash frozen in liquid nitrogen, and stored at -80°C.

Protein extraction and peptide preparation

Proteins were extracted by heating pelleted cells at 95°C for 10 min and gently shaking at room temperature for 30 min in a buffer solution (400 µL – 1760 µL; 50 mM HEPES pH 8.5 (Boston BioProducts #BB-2082), 1% SDS in HPLC grade water) before centrifuging at 14100 g for 20 min at room temperature and removing the supernatant. Sodium dodecyl sulfate (1%) is a strong detergent for diverse matrices including cell membranes (Hughes et al. 2014). Benzonase nuclease (50 units; Novagen #70746-3) was added to 400 µL extracted protein sample and incubated at 37°C for 30 min. Samples were reduced by adding 20 µL of 200 mM DTT (Fisher #BP172-5) in 50 mM HEPES pH 8.5 at 45°C for 30 min and alkylated with 40 µL of 400 mM iodoacetamide (Acros #122270050) in HEPES pH 8.5 for 30 min at 24°C. The reaction was quenched by adding 40 µL of 200 mM DTT in 50 mM HEPES pH 8.5. SpeedBead Magnetic Carboxylate Modified Particles (GE Healthcare #65152105050250 and #45152105050250) were prepared according to Hughes et al. (2014) and added (20 µg/µL) to 400 µL of extracted protein sample. Samples were incubated with formic acid (pH of 2-3) and washed with ethanol and acetonitrile using a magnetic rack. Protein was measured with the BCA method (Thermo Scientific Micro BCA Protein Assay Kit #23235) and digested overnight at 37°C with 1 part trypsin (Promega #V5280; dissolved in HEPES pH 8.0, 0.5 µg/µL) and 25 parts protein. Peptides were washed with acetonitrile and ethanol using a magnetic rack and diluted to a target concentration of 0.1% trifluoroacetic acid or 1% formic acid and a final concentration of 1 µg/µL.

Mass spectrometry of peptides

Similar to other analyses (Searle et al. 2018), peptides were analyzed using a Michrom Advance HPLC system coupled to a Q-Exactive mass spectrometer (Thermo Scientific instrument version 2.8) with a Michrom Advance CaptiveSpray source, using the constant injection concentration of 1 µg/µL (one microgram per microliter) to allow uniformity across the dataset. Samples were concentrated onto a C18 column (Reprosil-Gold, Dr. Maisch GmbH) and eluted in a non-linear, 200-min gradient of formic acid and acetonitrile buffers. Full MS1 scans were performed (35,000 resolution, 3e6 AGC target, 60 ms maximum IT, 385 to 1015 m/z) with overlapping DIA scans (17,500 resolution, 1e6 AGC target, 60 ms maximum IT, 24.0 m/z isolation windows, normalized collision energy of 27, loop count 25).

Proteomic data analysis

Data-independent acquisition mass spectrometry sample data were analyzed using Scaffold DIA (2.2.1), converted to mzML format (ProteoWizard 3.0.11748, Chambers et al (2012)), and individually searched against Syn8102_uniprot-proteome_UP000001422.fasta with a peptide and fragment mass tolerance of 10.0 ppm. Percolator (3.01) filtered peptides for a maximum false discovery rate of 0.01. Charged peptides (2-3) with length (6-30) were considered. EncyclopeDIA (0.9.6) selected the 5 highest quality fragment ions for quantitation (Searle et al. 2018).