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 \u00baC 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\u00b0C 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\u00b0C.
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Cells were lysed using a titanium sonicating micro-probe (20 sec, 10 repetitions) in a 6M urea and 50 \u03bcM ammonium bicarbonate solution. Disulfide bonds were reduced with dithiothreitol and alkylated with iodo-acetic acid. After additions of ammonium bicarbonate and methanol, 2 \u03bcg 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).
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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 \u03bcg 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 \u03bcm i.d. fused silica capillary column packed with C18 particles (Magic C18AQ, 100 \u00c5, 5 \u03bcm; Michrom, Bioresources, Inc., CA) fitted with a 2 cm long, 100 \u03bcm i.d. pre-column (Magic C18AQ, 200 \u00c5, 5\u03bcm; 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.
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Mass spectrometry was performed on replicates Av1 and Av2 independently using the Thermo Fisher (San Jose, Ca) linear ion trap \u2013Orbitrap (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.
Proteins identified in the Inferno hydrothermal vent plume meta-proteome (replicate Av1).\u00a0 Only proteins identified by peptides with a protein probability >0.9 are listed.\u00a0
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These data are reported as Supplementary Table 3 and discussed in Mattes et al., 2013. (doi:10.1038/ismej.2013.113)
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The FASTA information in the data was expanded to include the metadata when those FASTA headers were linked to GenBank.\u00a0
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Proteins that were identified in biological replicate Av2 that were not identified in biological replicate Av1. (GSO: Gamma Sulfur Oxidizer)
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![\"\"](\"https://datadocs.bco-dmo.org/d3/data_docs/SulfurOxidizers/Table5.png\")
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\"Although fewer proteins were identified in Av2, nearly all (94%) of the proteins identified in Av2 were also identified in Av1.\u00a0 Differences in the total number of proteins identified in replicate samples may result from differences in the amount of biomass obtained during sample processing.\"
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DMO notes:
\nPut multiple FASTA entries on separate lines
\nSplit out one number in FASTA header for linking
\nLeft it sorted by Total Independent Spectra column
\nAdded linkage column
\nRemoved commas in 'consensus annotation' column (signals database to put in new column)
\nReordered columns to put KEGG last -- much longer than any other column
Protein identifications
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Tandem mass spectra were interrogated against a composite database containing deduced protein sequences from lineages identified in the CTD17 clone library and lineages that are dominant in the deep ocean (background seawater). The database contained marine GSOs Candidatus Vesicomyosocius okutanii HA, Candidatus Ruthia magnifica Cm, the SUP05 metagenome (Walsh et al 2009), and SCGC AAA001-B15 (Arctic96BD-19 draft genome); the methylotrophs Methylobacter tundripaludum SV96 and Methylomicrobium alcaliphilum; iron-oxidizing bacteria Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1; abundant lineages in seawater Candidatus Pelagibacter ubique HTCC1062; Candidatus Pelagibacter ubique HTCC1002; Ammonia-oxidizing archaea Nitrosopumilus maritimus SCM1, an uncultured marine group II (Iverson et al 2012); an incomplete hydrothermal vent metagenome (Xie et al 2011); and common contaminants. SEQUEST (v. UW2011.01.1) was used to correlate observed tandem mass spectra to peptide sequence via theoretical tandem mass spectra from the composite database described above (Eng et al 1994, Eng et al 2008). For a detailed discussion of database considerations in community proteomics see Morris et al. (2010). SEQUEST parameters included a 3.75 Da peptide mass tolerance on MS1 spectra, specifying trypsin as the enzyme, variable oxidation modification on methionine (15.9949 Da), and static modification on Cysteine residues (57.021464 Da) resulting from alkylation.