Dataset: Nmarina targeted proteome

Cultivation conditions and sampling:
Cultures of the marine nitrite-oxidizing bacterium Nitrospira marina were grown under atmospheric and low oxygen concentrations (see Bayer et al 2020 for details). Cells were harvested for proteomic analysis during exponential growth. Each culture was mixed with an equal volume of RNALater and filtered by vacuum filtration onto 25 mm, 0.2 µm pore size Supor filters. Filters were frozen at -80°C until extraction.

Protein extraction and purification:
Samples were resuspended with 1800 µL of 1% SDS extraction buffer (1% SDS, 0.1M Tris/HCl pH 7.5, 10mM EDTA). Each sample was incubated at room temperature for 15 min, heated at 95C for 10 min, and shaken at room temperature (RT) at 350 rpm for 1 h. The protein extracts were decanted and centrifuged at 14100 x g for 20 min at RT. The supernatants were removed and concentrated by membrane centrifugation to approximately 300 µL in 5 K MWCO Vivaspin units (Sartorius Stedim, Goettingen, Germany). Each sample was precipitated with cold 50% methanol/50% acetone/0.5 mM HCl for 3 days at –20C, centrifuged at 14100 x g for 30 min at 4C, decanted and dried by vacuum concentration (Thermo Savant Speedvac) for 10 min or until dry. Pellets were resuspended in 1% SDS extraction buffer and left at RT for 1 h to completely dissolve. Total protein was quantified (Bio-Rad DC protein assay, Hercules, CA) with BSA as a standard.

Extracted proteins were purified from SDS detergent, reduced, alkylated and trypsin digested while embedded within a polyacrylamide tube gel, modified from a previously published method (Lu and Zhu et al 2005). A gel premix was made by combining 1 M Tris HCl (pH 7.5) and 40% Bis-acrylimide L 29:1 (Acros Organics) at a ratio of 1:3. The premix (103 µL) was combined with an extracted protein sample (35 µg-50 µg), TE Buffer, 7 µL 1% APS and 3 µL of TEMED (Acros Organics) to a final volume of 200 µL. After 1 h of polymerization at RT, 200 µL of gel fix solution (50% ETOH, 10% acetic acid in LC/MS grade water) was added to the top of the gel and incubated at RT for 20 min. Liquid was then removed and the tube gel was transferred into a new 1.5 mL microtube containing 1.2 mL of gel fix solution then incubated at RT, 350 rpm in a Thermomixer R (Eppendorf) for 1 h. The gel fix solution was then removed and replaced with 1.2 mL destaining solution (50% MeOH, 10% Acetic Acid in water) and incubated at 350rpm for 2h. Liquid was then removed, gel cut up into 1 mm cubes and then added back to tubes containing 1 mL of 50:50 acetonitrile:25 mM ammonium bicarbonate (ambic) incubated for 1 h, 350 rpm at RT. Liquid was removed and  replaced with fresh 50:50 acetonitrile:ambic and incubated at 16C at 350 rpm overnight. The above step was repeated for 1 h the following morning. Gel pieces were then dehydrated twice in 800 µL of acetonitrile for 10 min at room temperature and dried for 10 min in a ThermoSavant DNA110 speedvac after removing solvent. 600 µL of 10 mM DTT in 25 mM ambic was added to reduce proteins incubating at 56C, 350 rpm for 1 h. Unabsorbed DTT solution was then removed with volume measured. Gel pieces were washed with 25 mM ambic and 600 µL of 55 mM iodacetamide was added to alkylate proteins at RT, 350 rpm for 1 h. Gel cubes were then washed with 1 mL ambic for 20 min, 350 rpm at RT. Acetonitrile dehydrations and speedvac drying were repeated as above. Trypsin (Promega #V5280) was added in appropriate volume of 25 mM ambic to rehydrate and submerse gel pieces at a concentration of 1:20 µg trypsin:protein. Proteins were digested overnight at 350 rpm and 37C. Unabsorbed solution was removed and transferred to a new tube. 50 µL of peptide extraction buffer (50% acetonitrile, 5% formic acid in water) was added to gels, incubated for 20 min at RT then centrifuged at 14,100 x g for 2 min. Supernatant was collected and combined with unabsorbed solution. The above peptide extraction step was repeated combining all supernatants. Combined protein extracts were centrifuged at 14,100 x g for 20 min, supernatants transferred into a new tube and dehydrated down to approximately 10-20 µL in the speedvac. Concentrated peptides were then diluted in 2% acetonitrile 0.1% formic acid in water for storage until analysis. All water used in the tube gel digestion protocol was LC/MS grade, and all plastic microtubes were ethanol rinsed and dried prior to use.

Instruments:
Targeted analysis was conducted by mixing stable isotope labelled peptide standards that were produced using a plasmid inserted in E. coli containing the peptides of interest and calibrated against Pierce standard peptides (Pierce peptide calibration mixture 88321) to the samples and run in parallel reaction monitoring (PRM) mode on the Thermo Fusion mass spectrometer. Samples were injected onto a trap column (300 μm × 5 mm, 5-μm bead size, 100-Å pore size, C18 PepMap100, Thermo Scientific) at 10 μL/min and washed for 10 min with 0.1% formic acid in 2% acetonitrile. The trapped sample was then eluted at 500 nL/min onto a C18 column (100 μm × 150 mm, 3-μm particle size, 120-Å pore size, C18 Reprosil Gold, Dr. Maisch, packed in a New Objective PicoFrit column) with a 70-min nonlinear gradient (0.1% formic acid in water and 0.1% formic acid in 99.9% acetonitrile) into the mass spectrometer. PRM settings in the Fusion were set to 240,000 Orbitrap resolution at a scan range of 350–1,200 m/z for MS1, and a targeted mass list in the ion trap with collision-induced dissociation activation in normal scan rate mode with a maximum injection time of 35 ms for MS2 scans.