Seawater (~180 L) was collected from the stable hydrothermal vent plume issuing from the black smoker chimney Inferno (CTD17, 1 450 m). Whole water was transferred to clean 50 L polystyrene reservoirs and concentrated to ~230 ml with a Pellicon 2 tangential flow filtration system equipped with a 30 kDa Biomax Polyethersulfone cassette (Millipore Corporation, Billerica, MA) as described previously (Morris et al 2010). Cells were collected and concentrated in approximately 2 hours. Concentrated cells were flash frozen in liquid nitrogen and stored at -80 ºC until further processing at the University of Washington.Cell counts before and after filtration (6.9 x 1010 and 2.9 x 1010, respectively) indicate that we recovered 42% of the cells present in 180 L of hydrothermal vent plume water. Cells in the concentrated sample were divided into replicate samples (Av1 and Av2, ~115 ml each) and harvested by centrifuging at 4°C for 60 min (17,000 x g). The supernatant was discarded and cell pellets were rinsed with 100 uL of 20 mM Tris buffer pH 7.4 and stored -80°C.
Cells were lysed using a titanium sonicating micro-probe (20 sec, 10 repetitions) in a 6M urea and 50 μM ammonium bicarbonate solution. Disulfide bonds were reduced with dithiothreitol and alkylated with iodo-acetic acid. After additions of ammonium bicarbonate and methanol, 2 μg of sequence grade trypsin (Promega, Madison, WI) were added to each sample. Enzymatic digestions were incubated for 12 h at 37 oC. Resulting peptides were desalted using a macro-spin C18 column (NestGroup) following the manufacturers guidelines prior to analysis by mass spectrometry (MS).
Peptide concentrations from Axial volcano hydrothermal vent plume proteome replicates Av1 and Av2 were measured using the Thermo Scientific Nanodrop 2000/2000c, which measures the peptide bond absorbance at wavelength of 205 nm. Approximately 1 μg of peptide digest was used for each injection into the mass spectrometer. Each sample consisted of a complex mixture of peptides that were introduced into the mass spectrometer by reverse-phase chromatography using a brand new 15 cm long, 75 μm i.d. fused silica capillary column packed with C18 particles (Magic C18AQ, 100 Å, 5 μm; Michrom, Bioresources, Inc., CA) fitted with a 2 cm long, 100 μm i.d. pre-column (Magic C18AQ, 200 Å, 5μm; Michrom). Peptides were first trapped on the pre-column (5% ACN; 4 ml min-1; 7 min). Chromatographic separations were performed using an acidified (formic acid, 0.1% v/v) water-acetonitrile gradient (5-35% acetonitrile in 60 min) with a total run-time of 95 minutes.
Mass spectrometry was performed on replicates Av1 and Av2 independently using the Thermo Fisher (San Jose, Ca) linear ion trap –Orbitrap (LTQ-OT) hybrid tandem mass spectrometer. Peptides were analyzed using the data-independent Precursor Acquisition Independent from Ion Count (PAcIFIC) method (Panchaud et al 2009). Rather than requiring the mass spectrometer to select ions for fragmentation based on MS1 data, the PAcIFIC method systematically fragments ions at all m/z channels (Panchaud et al 2011). Each method file includes the full 95 minute linear HPLC gradient of 5-35% ACN over 60 minutes (see above) and covers a 21.5 m/z range using 14 contiguous, unique channels that span 2.5 m/z in the mass spectrometer. This results in a total of 45 method files per PAcIFIC analytical cycle to cover a full m/z range of 400-1400.
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