|Saito, Mak A.||Woods Hole Oceanographic Institution (WHOI)||Principal Investigator|
|Ake, Hannah||Woods Hole Oceanographic Institution (WHOI BCO-DMO)||BCO-DMO Data Manager|
|York, Amber D.||Woods Hole Oceanographic Institution (WHOI BCO-DMO)||BCO-DMO Data Manager|
FASTA file of protein identifications from the KM1128 in 2011. There is one entry for every protein ID.
Ocean metaproteomes were sampled and extracted as described in Saito et al., 2014. The protocols are reproduced here:
Samples were collected during the KM1128 METZYME research expedition (Metals and Enzymes in the Pacific) on the R/V Kilo Moana October 1-25, 2011 from Oahu, Hawaii to Apia, Samoa, with Carl Lamborg and Mak Saito as Chief Scientists. Microbial biomass for protein analyses was collected on vertical profiles using in situ high volume particle filtration pumps with a focus on the North Pacific and Equatorial regions. Protein samples were collected by a suite of 4 L/min and 8 L/min McLane Pumps (WTS-LV; McLane Research Laboratories Inc., Falmouth MA, USA) outfitted with custom Mini-MULVS multiple filter head systems. The 0.2-3.0 micron size fraction was collected on 142 mm filters (Supor, Pall Corp.) for analyses used in this study. The volume of water was pumped until a minimal flow rate was achieved or the allotted cast time period expired, typically ~300-500 L. Filters were sectioned immediately after pump retrieval, and protein samples (¼ filter) were stored in RNAlater reagent (Ambion, Life Technologies), which has been shown to be an effective preservative for cyanobacterial biomass (Saito et al., 2011), frozen at -80C, transported back to the laboratory on dry ice, and stored at -80C until analysis.
Total microbial protein (0.2-3.0um fraction) was extracted using detergent-based methods, described below. Total protein showed enhanced concentrations in the photic zone, particularly in the Equatorial and South Pacific portions of the transect (Fig. S1F). For protein extraction, samples were thawed and the filter and RNAlater preservative (Ambion Life Technologies) were separated. The removed preservative was spin-concentrated by a 5K MWCO membrane (Sartorius Stedim Biotech 6 mL, 5 K MWCO Vivaspin units; Goettingen, Germany), and rinsed with 0.1M Tris buffer to recover, desalt, and concentrate any suspended material. The sample filter was unfolded and placed in a larger tube to which 1% SDS extraction buffer (1% SDS, 0.1M Tris/HCL pH 7.5, 10mM EDTA) and the rinsed/desalted RNAlater fraction was added back. Each sample was incubated at room temperature for 15 minutes, heated at 95C for 10 minutes, and shaken at room temperature for 350 rpm for 1 h. The protein extract was decanted and placed in a new tube and centrifuged for 30 min x 3220 g at room temperature. The supernatants were removed and filtered through a 5 um low protein binding syringe filter (Fisher Scientific), and the filter rinsed with extraction buffer. The extracts were concentrated by 5 kD membrane centrifugation to a small volume, washed with extraction buffer, and concentrated again. Each sample was precipitated with cold 50% methanol (MeOH) 50% acetone 0.5 mM HCL for 3 days at –20C, centrifuged at 14100 x g (14500 rpm) for 30 m 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 room temperature (RT) for 1 h to redissolve. 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, using a modified protocol from a previously published method (38). The tube gel approach allowed all proteins including membrane proteins to be solubilized by detergent and purified while immobilized in the gel matrix. 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 ul) was combined with an extracted protein sample (usually 25 ug-200 ug), TE, 7 ul 1% APS and 3 ul of TEMED (Acros Organics) to a final volume of 200 ul. After 1 h of polymerization at RT, 200 ul 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 minutes. Liquid was then removed and the tube gel was transferred into a new 1.5 mL microtube.
|sequence||Precursors from all METZYME stations||unitless|
|scaffold_id||Links to the scaffold_id in the protein spectral counts dataset||unitless|
R/V Kilo Moana
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This is a MetZyme project cruise. The original cruise data are available from the NSF R2R data catalog.
MetZyme project researchers will determine the role of enzymatic activity in the cycling of trace metals. Specifically the research will address the following questions: (1) degradation of sinking particulate organic material in the Tropical North Pacific can be influenced by the ability of microbes to synthesize zinc proteases, which in turn is controlled by the abundance or availability of zinc, and (2) methylation of mercury is controlled, in part, by the activity of cobalt-containing enzymes, and therefore the supply of labile cobalt to the corrinoid-containing enzymes or co-factors responsible for methylation. To attain their goal, they will collect dissolved and particulate samples for trace metals and metalloenzymes from three stations along a biogeochemical gradient in the Tropical North Pacific (along 150 degrees West from 18 degrees North to the equator). Sinking particles from metal clean sediment traps will also be obtained. The samples will also be used to carry out shipboard incubation experiments using amendments of metals, metal-chelators, B12, and proteases to examine the sensitivity and metal limitation of heterotrophic, enzymatic degradation of organic matter within the oceanic "Twilight Zone" (100-500 m). This study will result in a novel metaproteomic/metalloenzyme datasets that should provide insights into the biogeochemical cycling of metals, as well as co-limitation of primary productivity and controls on the export of carbon from the photic zone. In addition to the final data being contributed to BCO-DMO, an online metaproteomic data server will be created so the community has access to the raw data files generated by this research.
GEOTRACES gained momentum following a special symposium, S02: Biogeochemical cycling of trace elements and isotopes in the ocean and applications to constrain contemporary marine processes (GEOSECS II), at a 2003 Goldschmidt meeting convened in Japan. The GEOSECS II acronym referred to the Geochemical Ocean Section Studies To determine full water column distributions of selected trace elements and isotopes, including their concentration, chemical speciation, and physical form, along a sufficient number of sections in each ocean basin to establish the principal relationships between these distributions and with more traditional hydrographic parameters;
* To evaluate the sources, sinks, and internal cycling of these species and thereby characterize more completely the physical, chemical and biological processes regulating their distributions, and the sensitivity of these processes to global change; and
* To understand the processes that control the concentrations of geochemical species used for proxies of the past environment, both in the water column and in the substrates that reflect the water column.
GEOTRACES will be global in scope, consisting of ocean sections complemented by regional process studies. Sections and process studies will combine fieldwork, laboratory experiments and modelling. Beyond realizing the scientific objectives identified above, a natural outcome of this work will be to build a community of marine scientists who understand the processes regulating trace element cycles sufficiently well to exploit this knowledge reliably in future interdisciplinary studies.
Expand "Projects" below for information about and data resulting from individual US GEOTRACES research projects.
|NSF Division of Ocean Sciences (NSF OCE)|
|Gordon and Betty Moore Foundation: Marine Microbiology Initiative (MMI)|