Dataset: BB2-AT2 Cytosolic Metallome

Location: All physiological work within this dataset was conducted at Woods Hole Oceanographic Institution on a strain of Pseudoalteromonas collected from Scripps Pier, California coast in 1995.

Methodology:

Referenced from Mazzotta, et al. in press:
Marine broth medium was prepared by microwave sterilization49 of 800 mL of 0.2 µm-filtered Vineyard Sound seawater with the addition of 0.2 µm-filter-sterilized solutions of 4 g peptone (Fisher Scientific) and 0.8 g yeast extract (BD Difco). Marine agar plates were prepared with the same composition as that of the marine broth described herein, with the inclusion of 12 g granulated agar (Fisher Scientific) and sterilization achieved by autoclaving for 30 min. Cultures of Pseudoalteromonas sp. BB2-AT2 were revived from a 20% glycerol stock of BB2-AT2 (provided by Kay Bidle, Rutgers University) and used to inoculate approximately 1 mL of marine broth. The inoculated solution was allowed to incubate at 23 °C for 3 hours, then streaked onto a marine agar plate with 50 µL inoculum and incubated at 23 °C overnight. Pellets of BB2-AT2 were grown in volume 500 mL of marine broth medium by inoculation at 23 °C with selection of a single colony from a marine agar plate. Growth curves were obtained with a SpectraMax Me5 unit (Molecular Devices) through absorbance measurements at 600 nm pipetting aliquots into a Corning clear 96 well plate at each time point. Samples were analyzed at 23 °C with a kinetic run over a period of 18 hours with a read interval of 30 minutes, with aliquots added at every timepoint. To validate this approach these plate reader growth rates were successfully intercompared with growth measurements using a Shimadzu UV-1601 spectrophotometer in a quartz cuvette (b = 1 cm) with a 2.5 mL aliquot of inoculated medium (Supplementary Information). Pellets were harvested in mid-exponential phase by centrifugation at 8,000 rpm for 20 minutes using an Eppendorf 5810R centrifuge at 3 °C, the solution was decanted, and the pellet washed with 0.2 µm-filtered seawater (4 mL x 3). Pellets of biomass

For metalloproteomic analysis, all plasticware used was acid-washed unless otherwise mentioned. Buffers were prepared in Milli-Q water with Tris base (Fisher BioReagents) and NaCl (Sigma Aldrich, trace metal basis) with removal of extraneous metal contaminants from solution by Chelex prepared accordingly.50 The pellet of BB2-AT2 was suspended in approximately 6 mL of 50 mM Tris buffer (pH 8.8) under anaerobic conditions (<1 ppm O2) in an N2–filled anaerobic chamber (Coy Laboratory Products Inc.) using a Fisherbrand Model 120 sonic dismembrator, sonicated for 40 s at 2s on/off intervals while on ice. The resulting solution was then diluted to approximately 30 mL with additional 50 mM Tris buffer (pH 8.8) and centrifuged at 4,000 rpm at 3 °C for 15-20 min. The resulting supernatant was decanted and then purified by anion exchange chromatography using an Agilent 1100 series HPLC. A single-use GE HiTrap Q HP anion exchange column was conditioned prior to use by washing the column with 50 mM Tris buffer (pH 8.8) for 5 min at a flow rate of 1 mL/min, then 50 mM Tris buffer containing 1 M sodium chloride (pH 8.8) for 5 min at a flow rate of 1 mL/min and finally with 50 mM Tris buffer (pH 8.8) for 5 min at a flow rate of 1 mL/min. The supernatant was then loaded onto the column via a 60 mL loading syringe onto the column using Carter 12/8 cassette peristaltic pump at a rate of 0.5 mL/min. The sample-loaded column was then connected to the Agilent HPLC system and eluted with a gradient of sodium chloride at a rate of 0.25 mL/min. The method includes the following gradient- 50 mM Tris buffer (pH 8.8) for 5 minutes, followed by a linear increase from 100% 50 mM Tris buffer (pH 8.8) to 100% 50 mM Tris buffer containing 1 M NaCl (pH 8.8) over the course of 40 min and then 50 mM Tris buffer (pH 8.8, 1 M NaCl) for 8 min. Fractions were collected with a Bio-Rad 2110 fraction collector in 2 mL microtubes at a rate of 1 fraction every 4 min, and immediately stored on ice. Anion exchange fractions were concentrated prior to separation by size exclusion chromatography. 5000 Da molecular weight cutoff PES Vivaspin columns were washed with 0.5 mL (x 2) Tris pH 8.8 buffer prior to use. Anion exchange fractions were loaded onto the Vivaspin columns and centrifuged at 5000 rpm for 30 min using an Eppendorf MiniSpin. 0.1 mL of each concentrated anion exchange fraction was injected into a Michrom BioResources, Inc. HPLC system containing a Tosoh Bioscience 5-µm particle size TSKgel G3000SWxl size exclusion column with guard column. Samples were eluted using an isocratic method of 50 mM Tris buffer (pH 7.5, 50 mM sodium chloride) at a flow rate of 0.5 mL/min. Fractions from size exclusion chromatography were collected using a CTC Analytics HTC Pal autosampler system into an acid-washed 1.2 mL 96-well storage plate (Thermo Scientific), covered for storage until analysis with an acid-washed 96 cap sealing mat (Thermo Scientific). For protein digestion, 90 μL of each sample was diluted with 96 μL 100 mM ammonium bicarbonate solution and 10 μL acetonitrile. Then 15 μL of a freshly-prepared 10 mM dithiothreitol solution was added to the samples and incubated at 70 °C for 30 min with shaking at 400 rpm. The samples were then allowed to cool to room temperature, and 30 μL freshly-prepared 20 mM iodoacetamide was added to each sample and allowed to incubate for 30 min in the dark. Following reduction and alkylation, 10 μL of a 0.03 μg/μL trypsin stock (Promega Gold) was added to each sample and then incubated for approximately 18 hours at 37 °C with shaking at 400 rpm. Samples were then analyzed by an LC-MS on a Q-Exactive (Thermo Scientific) after separation on a C18 column following a gradient of 2-95% acetonitrile over a 90 min gradient using a Michrom Paradigm MG4 liquid chromatography system. Samples were ionized by electrospray with 1800 mV potential. Proteomic analyses were used for peptide spectra matching using the SEQUEST algorithm within Proteome Discoverer (v. 2.1) and visualized in Scaffold (v. 4); protein threshold = 99.9%; minimum # peptides = 2; peptide threshold = 99%.

Instruments:

All work performed in N2–filled anaerobic chamber (Coy Laboratory Products Inc.).
Anion exchange chromatography: Agilent 1100 series HPLC.
Size exclusion chromatography: Michrom BioResources, Inc. HPLC system containing a Tosoh Bioscience 5-µm particle size TSKgel G3000SWxl size exclusion column with guard column.
Samples were then analyzed by an LC-MS on a Q-Exactive (Thermo Scientific) using a Michrom Paradigm MG4 liquid chromatography system.
Metals analyzed on iCAP-Q inductively coupled plasma-mass spectrometer (Thermo) with an SC-4 DX FAST autosampler (Elemental Scientific, Inc.).