http://lod.bco-dmo.org/id/dataset/718886
eng; USA
utf8
dataset
Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2017-11-08
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Shotgun Proteomics of Bering Sea algal incubations.
2017-11-08
publication
2017-11-08
revision
BCO-DMO Linked Data URI
2017-11-08
creation
http://lod.bco-dmo.org/id/dataset/718886
Brook L. Nunn
University of Washington
principalInvestigator
Rodger Harvey
Old Dominion University
principalInvestigator
William S. Noble
University of Washington
principalInvestigator
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
publisher
Cite this dataset as: Nunn, B. L., Noble, W. S., Harvey, R. (2017) Shotgun Proteomics of Bering Sea algal incubations. Biological and Chemical Oceanography Data Management Office (BCO-DMO). Version Date 2017-11-08 [if applicable, indicate subset used]. http://lod.bco-dmo.org/id/dataset/718886 [access date]
Dataset Description: <p>Bering Sea water was collected during Bering Sea Ecosystem Study (BEST; Wiese et al. 2012) cruises on the outer shelf during the spring of 2009 (59.9037°N, 176.1278°W; sampling depth 5 m; water column depth 136 m) and 2010 (56.7272° N, 169.4271°W; sampling depth 36 m; water column depth 104 m) in areas which coincided with the developing spring bloom adjacent to the retreating ice.&nbsp;</p>
<p>Cruise: BEST Cruise 2012 (Bering Sea Ecosystem Study)</p>
<p>Water was collected from ship trap samples, filtered, and algal fractions were lysed, digested and analyzed using proteomic mass spectrometry.<br />
<br />
Data are available for download at the EBI PRIDE Archive. Project number: PXD006417 </p>
<p>Homepage:&nbsp;<a href="https://www.ebi.ac.uk/pride/archive" target="_blank">https://www.ebi.ac.uk/pride/archive</a><br />
Project URL:&nbsp;<a href="https://www.ebi.ac.uk/pride/archive/projects/PXD006417" target="_blank">https://www.ebi.ac.uk/pride/archive/projects/PXD006417</a><br />
Data URL:&nbsp;<a href="https://www.ebi.ac.uk/pride/archive/projects/PXD006417/files" target="_blank">https://www.ebi.ac.uk/pride/archive/projects/PXD006417/files</a></p>
<p>Data are published in&nbsp;Moore, E.K., Faux, J., Goodlett, D.R., Harvey, H.R., and Nunn, B.L. 2014. Protein recycling in Bering Sea algal incubations. Marine Ecology Progress Series. 515:45-59. DOI:&nbsp;<a href="http://doi.org/10.3354/meps10936" target="_blank">10.3354/meps10936</a></p> Methods and Sampling: <p>Bering Sea water was collected during Bering Sea Ecosystem Study (BEST; Wiese et al. 2012) cruises on the outer shelf during the spring of 2009 (59.9037°N, 176.1278°W; sampling depth 5 m; water column depth 136 m) and 2010 (56.7272° N, 169.4271°W; sampling depth 36 m; water column depth 104 m) in areas which coincided with the developing spring bloom adjacent to the retreating ice. The phytoplankton community at the time of sampling was diatom dominated (Lomas et al. 2012, Moran et al. 2012). In each year, single 20 l carboys were filled from the CTD rosette taken from the chlorophyll maximum based on chlorophyll fluorescence at the time of sampling. In order to increase the amount of algal material for analysis throughout the incubation, 1 l of concentrated phytoplankton material was obtained by gently passing 10 l of CTD water from an additional bottle through a 10 µm mesh and combined with untreated seawater to make up the 20 l incubation. Macrozooplankton were excluded from the incubation by passing CTD water through a 1 mm plankton net before being added to the 20 l carboys.<br />
<br />
Incubations were placed in shipboard −1°C cold rooms for 11 d (2009) and 53 d (2010) for the duration of the experimental period. Carboys were covered to exclude light throughout the incubations and aerated with filtered air. At regular time points, carboys were gently mixed until algal material was homogeneous - ly distributed, and 1 l water samples were collected and filtered onto 25 mm pre-combusted glass fiber filters (GF/F) and 37 mm polycarbonate (0.2 µm) filters for analysis (Table 1). In addition, whole water samples were collected at each time point and filtered onto 0.2 µm filters, DAPI stained, and fixed onto microscope slides for bacterial counts. All incubation particles and bacterial slides were stored at −70°C until analysis or counting. Stained bacterial cells were counted on an Olympus BH2-RFCA fluorescent microscope.<br />
<br />
Total hydrolyzable amino acids (THAAs) were identified and quantified by gas chromatography/ mass spectrometry (GC/MS) using the EZFaast method (Phenomonex), which uses derivatization of amino acids with propyl chloroformate and propanol for detection (Waldhier et al. 2010). Briefly, suspended particles collected on GF/Fs were hydrolyzed for 4 h at 110°C (Cowie &amp; Hedges 1992) with 6 M ana lyticalgrade HCl and L-γ-Methylleucine as the recovery standard. Following hydrolyzis and derivatization, amino acids were quantified using an Agilent 6890 capillary GC with samples injected at 250°C and separated on a DB-5MS (0.25 mm ID, 30 m) column with H2 as the carrier gas. The oven was ramped from an initial temperature of 110°C to 280°C at 10°C min−1 followed by a 5 min hold. Amino acid identification was accomplished by an Agilent 5973N mass spectrometer run under the same conditions with helium as the carrier gas and mass spectral acquisition over the 50 to 600 Da range. The protein bovine serum albumin (BSA) was analyzed in parallel to correct for responses among individual amino acids and calculation of molar ratios. The analytical precision (% relative standard deviation) for amino acid analysis was ±5%. Amino acids were normalized to percent carbon and nitrogen present in bulk samples analyzed by standard combustion methods. Total protein content was also estimated by the Bradford assay (Bradford 1976).<br />
<br />
Incubation particles collected on polycarbonate filters were extracted for proteins with pulse sonication in 6 M urea with a Branson 250 sonication probe at 20 kHz for 30 s on ice. The extracts were then frozen at −80°C, thawed, and sonicated again for 30 s on ice. This was repeated for a total of 5 sonications and 4 freeze/thaw cycles. Filter extracts of each incubation time point were then digested in 3 replicate groups: (1) standard tryptic digestion with reduction and alkylation (Nunn et al. 2010); (2) digestion with Endoproteinase GluC (Endo GluC), which cleaves peptide bonds C-terminal to glutamic acid (Drapeau et al. 1972) and to a lesser extent aspartic acid (Birktoft &amp; Breddam 1994), to increase the number of proteins identified; and (3), incubation with Peptide N-Glycosidase F (PNGase F), which hydrolyzes nearly all types of N-glycan chains from glycoproteins and glycopeptides (Maley et al. 1989), in order to observe potentially modified proteins prior to tryptic digestion. All digests were concentrated using a speedvac to a volume that gave a final protein concentration of 1 µg per 10 µl based on measured protein concentrations of filter extracts. The uniform 1 µg per 10 µl protein concentration ensured that results would not be biased by sampling, or protein concentration differences at different incubation time points.</p>
<p>Protein identification of sample digests was performed via shotgun proteomic tandem mass spectrometry (MS2; Aebersold &amp; Mann 2003). Digests were analyzed using full scan (specific mass to charge ratio [m/z] 350−2000), followed by gas phase fractionation with repeat analyses over multiple narrow, but overlapping, m/z ranges (Yi et al. 2002, Nunn et al. 2006). Mass spectra were evaluated and database searched with an in-house copy of SEQUEST (Eng et al. 1994, 2008). All searches were performed with no assumption of proteolytic enzyme cleavage (e.g. trypsin, Endo GluC) to allow for identification of protein degradation products due to microbial recycling. A fixed modification was set for 57 Da on cysteine and a variable modification of 16 Da on methionine resulting from alkylation and reduction steps, respectively. A variable 1 Da modification was set for asparagine on PNGase F + trypsin digested samples to account for the conversion of asparagine to aspartic acid after cleavage of glycan chains with the use of PNGase F (Plummer et al. 1984), which takes place specifically at the consensus sequence Asn-XxxSer/Thr where Xxx can be any amino acid except proline (Bause &amp; Hettkamp 1979).</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1233589 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1233589
onGoing
Brook L. Nunn
University of Washington
206-616-9023
Department of Genome Sciences, Foege Building S113 3720 15th Ave NE
Seattle
WA
98195
USA
brookh@uw.edu
pointOfContact
Rodger Harvey
Old Dominion University
757-683-5657
4600 Elkhorn Ave
Norfolk
VA
23508
US
rharvey@odu.edu
pointOfContact
William S. Noble
University of Washington
Department of Genome Sciences Foege Building S113, 3720 15th Ave NE
Seattle
WA
98195
USA
wnoble@uw.edu
pointOfContact
asNeeded
Unknown
Repository
Project
Project_URL
Agilent 6890 capillary GC
Agilent 5973N
theme
None, User defined
No BCO-DMO term
project
external_link
featureType
BCO-DMO Standard Parameters
CTD - profiler
Fluorescence Microscope
Gas Chromatograph
Mass Spectrometer
instrument
BCO-DMO Standard Instruments
otherRestrictions
otherRestrictions
Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use.
Collaborative Research: Linking geochemistry and proteomics to reveal the impact of bacteria on protein cycling in the ocean
https://osprey.bco-dmo.org/project/675324
Collaborative Research: Linking geochemistry and proteomics to reveal the impact of bacteria on protein cycling in the ocean
<p><em>Text from NSF</em><em> award abstract:</em></p>
<p>Although proteins represent the primary source of new organic nitrogen in the ocean, the identification of individual proteins and mechanisms modulating their preservation has faced analytical and computational challenges in deciphering the vast suite of possible sequences and degradation by-products. Recent efforts to link geochemical cycling, biomedical proteomics and bioinformatics has demonstrated that only a small subset of the suite of proteins produced by marine diatoms appear to survive the degradation process, and those that do are largely protected by physical and enthalpic barriers to microbial attack. Although these discoveries help to explain the survival of individual proteins, they also generate multiple questions regarding bacteria as the dominant recyclers of organic nitrogen and carbon and needs for specific approaches to characterize modified protein products. Bacteria dominate the water column and sedimentary systems in both numbers and diversity, yet their relative contribution to the preserved proteomic pool appears low.</p>
<p>In this project, researchers at Old Dominion Universityand the University of Washington will join forces to decipher the bacterial role in protein recycling and their potential contribution. By integrating high mass accuracy tandem mass spectrometry-based proteomics with stable isotope-based geochemical analysis, they hope to identify those bacterial proteins initially synthesized during organic matter recycling. Three research objectives drive this investigation: (1) to determine the potential contribution of bacteria proteins to marine organic matter; (2) to identify those protein(s) synthesized by heterotrophic marine bacteria during initial stages of organic matter degradation; (3) to determine if glycan (carbohydrate) modifications represent an important component of preserved, yet unidentified, peptides seen in our analysis of oceanic particles and sediments.</p>
<p>Broader Impacts: This project will provide multiple opportunities for interdisciplinary student training in marine chemistry and proteomics as well as address the goal of disseminating results and tools to a broad audience. In the more traditional role, this project will expand the career for a female principal investigator in marine proteomics, support both graduate and undergraduate students at ODU which include opportunities for minority enrichment and provide training for a postdoctoral fellow at UW. On the broader level, the ODU PI participates in high school outreach programs for high achieving students in the local school which provides for summer internships and enrichment programs.</p>
<p><strong>Relevant Links:</strong></p>
<p>Old Dominion University: <a href="http://mogel.cbl.umces.edu/" target="_blank">Marine Organic Geochemistry and Ecology Laboratory (MOGEL) Lab Website</a><br />
Bering Sea Ecosystem Study: <a href="https://www.eol.ucar.edu/projects/best/" target="_blank">Data Archive</a><br />
Environmental Proteomics: <a href="http://www.environmentalproteomics.org/bacterial-degradation-of-proteins.html" target="_blank">Bacteria Recyclers in the Ocean</a><br />
Environmental Proteomics: <a href="http://www.environmentalproteomics.org/proteomics-at-subzero-temperatures.html" target="_blank">Proteomics of Colwellia psychretheca at subzero temperatures </a></p>
Bacterial Recyclers
largerWorkCitation
project
eng; USA
oceans
-176.1278
-176.1278
59.9037
59.9037
2017-11-08
0
BCO-DMO catalogue of parameters from Shotgun Proteomics of Bering Sea algal incubations.
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
http://lod.bco-dmo.org/id/dataset-parameter/719758.rdf
Name: Repository
Units: unitless
Description: <p>Name of database where data are currently served</p>
http://lod.bco-dmo.org/id/dataset-parameter/719759.rdf
Name: Project
Units: unitless
Description: <p>Unique project identifier for the database where data are currently served</p>
http://lod.bco-dmo.org/id/dataset-parameter/719760.rdf
Name: Project_URL
Units: unitless
Description: <p>Link to project page at the database where data are currently served</p>
GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
193
https://datadocs.bco-dmo.org/file/B11vANAil1VP11/BeringSt_Algal_Incubations_Moore.csv
BeringSt_Algal_Incubations_Moore.csv
Primary data file for dataset ID 718886
download
https://osprey.bco-dmo.org/dataset/718886/data/download
download
onLine
dataset
<p>Bering Sea water was collected during Bering Sea Ecosystem Study (BEST; Wiese et al. 2012) cruises on the outer shelf during the spring of 2009 (59.9037°N, 176.1278°W; sampling depth 5 m; water column depth 136 m) and 2010 (56.7272° N, 169.4271°W; sampling depth 36 m; water column depth 104 m) in areas which coincided with the developing spring bloom adjacent to the retreating ice. The phytoplankton community at the time of sampling was diatom dominated (Lomas et al. 2012, Moran et al. 2012). In each year, single 20 l carboys were filled from the CTD rosette taken from the chlorophyll maximum based on chlorophyll fluorescence at the time of sampling. In order to increase the amount of algal material for analysis throughout the incubation, 1 l of concentrated phytoplankton material was obtained by gently passing 10 l of CTD water from an additional bottle through a 10 µm mesh and combined with untreated seawater to make up the 20 l incubation. Macrozooplankton were excluded from the incubation by passing CTD water through a 1 mm plankton net before being added to the 20 l carboys.<br />
<br />
Incubations were placed in shipboard −1°C cold rooms for 11 d (2009) and 53 d (2010) for the duration of the experimental period. Carboys were covered to exclude light throughout the incubations and aerated with filtered air. At regular time points, carboys were gently mixed until algal material was homogeneous - ly distributed, and 1 l water samples were collected and filtered onto 25 mm pre-combusted glass fiber filters (GF/F) and 37 mm polycarbonate (0.2 µm) filters for analysis (Table 1). In addition, whole water samples were collected at each time point and filtered onto 0.2 µm filters, DAPI stained, and fixed onto microscope slides for bacterial counts. All incubation particles and bacterial slides were stored at −70°C until analysis or counting. Stained bacterial cells were counted on an Olympus BH2-RFCA fluorescent microscope.<br />
<br />
Total hydrolyzable amino acids (THAAs) were identified and quantified by gas chromatography/ mass spectrometry (GC/MS) using the EZFaast method (Phenomonex), which uses derivatization of amino acids with propyl chloroformate and propanol for detection (Waldhier et al. 2010). Briefly, suspended particles collected on GF/Fs were hydrolyzed for 4 h at 110°C (Cowie &amp; Hedges 1992) with 6 M ana lyticalgrade HCl and L-γ-Methylleucine as the recovery standard. Following hydrolyzis and derivatization, amino acids were quantified using an Agilent 6890 capillary GC with samples injected at 250°C and separated on a DB-5MS (0.25 mm ID, 30 m) column with H2 as the carrier gas. The oven was ramped from an initial temperature of 110°C to 280°C at 10°C min−1 followed by a 5 min hold. Amino acid identification was accomplished by an Agilent 5973N mass spectrometer run under the same conditions with helium as the carrier gas and mass spectral acquisition over the 50 to 600 Da range. The protein bovine serum albumin (BSA) was analyzed in parallel to correct for responses among individual amino acids and calculation of molar ratios. The analytical precision (% relative standard deviation) for amino acid analysis was ±5%. Amino acids were normalized to percent carbon and nitrogen present in bulk samples analyzed by standard combustion methods. Total protein content was also estimated by the Bradford assay (Bradford 1976).<br />
<br />
Incubation particles collected on polycarbonate filters were extracted for proteins with pulse sonication in 6 M urea with a Branson 250 sonication probe at 20 kHz for 30 s on ice. The extracts were then frozen at −80°C, thawed, and sonicated again for 30 s on ice. This was repeated for a total of 5 sonications and 4 freeze/thaw cycles. Filter extracts of each incubation time point were then digested in 3 replicate groups: (1) standard tryptic digestion with reduction and alkylation (Nunn et al. 2010); (2) digestion with Endoproteinase GluC (Endo GluC), which cleaves peptide bonds C-terminal to glutamic acid (Drapeau et al. 1972) and to a lesser extent aspartic acid (Birktoft &amp; Breddam 1994), to increase the number of proteins identified; and (3), incubation with Peptide N-Glycosidase F (PNGase F), which hydrolyzes nearly all types of N-glycan chains from glycoproteins and glycopeptides (Maley et al. 1989), in order to observe potentially modified proteins prior to tryptic digestion. All digests were concentrated using a speedvac to a volume that gave a final protein concentration of 1 µg per 10 µl based on measured protein concentrations of filter extracts. The uniform 1 µg per 10 µl protein concentration ensured that results would not be biased by sampling, or protein concentration differences at different incubation time points.</p>
<p>Protein identification of sample digests was performed via shotgun proteomic tandem mass spectrometry (MS2; Aebersold &amp; Mann 2003). Digests were analyzed using full scan (specific mass to charge ratio [m/z] 350−2000), followed by gas phase fractionation with repeat analyses over multiple narrow, but overlapping, m/z ranges (Yi et al. 2002, Nunn et al. 2006). Mass spectra were evaluated and database searched with an in-house copy of SEQUEST (Eng et al. 1994, 2008). All searches were performed with no assumption of proteolytic enzyme cleavage (e.g. trypsin, Endo GluC) to allow for identification of protein degradation products due to microbial recycling. A fixed modification was set for 57 Da on cysteine and a variable modification of 16 Da on methionine resulting from alkylation and reduction steps, respectively. A variable 1 Da modification was set for asparagine on PNGase F + trypsin digested samples to account for the conversion of asparagine to aspartic acid after cleavage of glycan chains with the use of PNGase F (Plummer et al. 1984), which takes place specifically at the consensus sequence Asn-XxxSer/Thr where Xxx can be any amino acid except proline (Bause &amp; Hettkamp 1979).</p>
Specified by the Principal Investigator(s)
asNeeded
7.x-1.1
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
PI Supplied Instrument Name: Instrument Name: CTD - profiler Instrument Short Name: Instrument Description: The Conductivity, Temperature, Depth (CTD) unit is an integrated instrument package designed to measure the conductivity, temperature, and pressure (depth) of the water column. The instrument is lowered via cable through the water column. It permits scientists to observe the physical properties in real-time via a conducting cable, which is typically connected to a CTD to a deck unit and computer on a ship. The CTD is often configured with additional optional sensors including fluorometers, transmissometers and/or radiometers. It is often combined with a Rosette of water sampling bottles (e.g. Niskin, GO-FLO) for collecting discrete water samples during the cast.
This term applies to profiling CTDs. For fixed CTDs, see https://www.bco-dmo.org/instrument/869934. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/130/
PI Supplied Instrument Name: Instrument Name: Fluorescence Microscope Instrument Short Name: Instrument Description: Instruments that generate enlarged images of samples using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption of visible light. Includes conventional and inverted instruments. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB06/
Agilent 6890 capillary GC
Agilent 6890 capillary GC
PI Supplied Instrument Name: Agilent 6890 capillary GC Instrument Name: Gas Chromatograph Instrument Short Name:Gas Chromatograph Instrument Description: Instrument separating gases, volatile substances, or substances dissolved in a volatile solvent by transporting an inert gas through a column packed with a sorbent to a detector for assay. (from SeaDataNet, BODC) Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB02/
Agilent 5973N
Agilent 5973N
PI Supplied Instrument Name: Agilent 5973N Instrument Name: Mass Spectrometer Instrument Short Name:Mass Spec Instrument Description: General term for instruments used to measure the mass-to-charge ratio of ions; generally used to find the composition of a sample by generating a mass spectrum representing the masses of sample components. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB16/