Dataset: Protein Abundance data from Zn and CO2 culture experiments on the diatom Phaeodactylum tricornutum

Proteomic Analysis: "Harvested cell pellets were removed from the freezer, resuspended in lysis buffer (2% SDS, 50mM TEAB pH 8.5), and incubated at 95°C and mixed at 350 rmp for 10 (eppendorf; Thermomixer R). The samples were cooled on ice following the heated lysis buffer treatment, and 2 μL of benzonase nuclease (MilliporeSigma 707463) was added to each. This solution was left to incubate at 37°C for 30 min, shaking at 350 rpm. The extract was centrifuged at 14,100 × g for 10 minutes to remove cellular debris, and the extracted proteins in the supernatant were transferred to an ethanol washed tube. After the sample protein extraction, the total protein was quantified using a colorimetric assay at 562 nm (Pierce BCA total protein assay, Thermo Scientific 23225). Samples containing 25 μg of total protein were each subsampled into 200 µL of lysis buffer for the reduction, alkylation, and digestion of proteins.

To alkylate the samples, 4 µL of 500 mM dithiothreitol (DTT; Thermo Fisher Scientific 12-3-3483) in 50 mM ammonium bicarbonate (Ambic; Thermo Fisher Scientific 1066-33-7) was added and incubated at 45°C for 30 minutes. The alkylation was completed with an addition of 12 µL 500mM iodoacetamide (MilliporeSigma 144-48-9) in 50 mM Ambic, which incubated in the dark for 30 min at room temperature. Finally, the reaction was quenched with a second addition of 4 µL of DTT with a 10 min incubation and acidification using 12% phosphoric acid. Following extraction, reduction, alkylation, and acidification, the samples were transferred to an S-trap column (Protifi, S-Trap Mini Column) to clean, isolate, digest, and elute the proteins for analysis. Briefly, samples were thoroughly washed with binding buffer and a final wash with 90% methanol, digested with trypsin at a ratio of 25 µg protein:1 µg trypsin, left to incubate overnight at 37°C, and eluted with 50 mM Ambic, 50% formic acid, and 50% acetonitrile (ACN, Thermo Fisher Scientific A955-4) in 0.2% formic acid. This volume was quantified, and the purified protein was quantified using the BCA colorimetric assay.

Tryptic peptides were analyzed with liquid chromatography coupled with tandem mass spectrometry (LS/MS/MS) where a Thermo Vanquish Neo HPLC reverse phase chromatography system was paired with a Thermo Scientific Fusion mass spectrometer using a Thermo Flex nanospray source. The peptides were eluted on a reverse phase C18 column (0.1 x 500 mm ID, 2 µm particle size, 120 Å pore size, C18 Reprosil-Gold, Dr. Maisch GmbH) using a 500 nl/min flow rate. All solvents were prepared with Fisher Optima grade reagents. Liquid chromatography was a nonlinear 60-minute gradient from 5%-95% buffer B, where A was 0.1% formic acid in water and B was 0.1% formic acid in ACN. Data independent acquisition (DIA) was used for analysis of the biological triplicates. The DIA scans comprised a precursor range of 380 m/z to 980 m/z on the Orbitrap with 24 m/z MS2 non-overlapping windows. The MS2 scans ranged from 150 to 2000 m/z, and the Orbitrap resolution was 60K for MS1 and 30K for MS2.

The generated mass spectra from DIA analysis were searched against the translated genomes obtained from NCBI using MSFragger within FragPipe (v22.0: https://github.com/Nesvilab/FragPipe). An initial precursor mass tolerance of -10 to 10 ppm, a fragment mass tolerance of 10 ppm, a parent tolerance of 10.0 ppm, and a +57 carbamidomethyl fixed modification of cysteine were used, and MSBooster rescoring was used. Variable modifications were a +16 oxidation modification on methionine and a +42 acetyl modification on the N-terminus. Only peptides of length 6 to 50 and precursor charge states 2 to 4 were included, and a maximum of 1 missed cleavage was allowed. False discovery rates were calculated using decoys generated from a reverse database. A spectral library was generated from MSFragger, and DIA quantification was performed using DIA-NN to obtain a maximum protein false discovery rate of 0.01 (1%)."