http://lod.bco-dmo.org/id/dataset/958552
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
2025-04-10
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Metaproteomics analyses of mixed-layer water from Hawaii Ocean Time-series Station ALOHA, collected on R/V Kilo Moana cruise KM2204 in March 2022, and incubated with 15N2 to track biosynthetic incorporation by diazotrophic microbes
2025-04-10
publication
2025-04-10
revision
BCO-DMO Linked Data URI
2025-04-10
creation
http://lod.bco-dmo.org/id/dataset/958552
Jacob Waldbauer
University of Chicago
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
documentDigital
Cite this dataset as: Waldbauer, J. (2024) MassIVE MSV000096410 - HOT335 15N2-tracking Metaproteomics. MassIVE. (Version 1) Version Date 2024 [if applicable, indicate subset used]. doi:10.25345/C5C24R05N [access date]
HOT335 15N2 Incubations Dataset Description: Methods and Sampling: <p>Mixed-layer water (25 meters (m) depth) was sampled at Station ALOHA using 12-liter (L) Niskin bottles on the HOT CTD rosette. To prepare 15N₂-enriched seawater for incubations, water was filtered first through nitex mesh (250-micrometers (µm)) and then through 0.2 µm-pore filters (Millipak) into pre-cleaned 2L pyrex glass bottles with gas-port caps (Chemglass). Water was then degassed under vacuum with vigorous stirring (Velp MST magnetic stir plates) for at least 30 minutes. The evacuated headspace was then filled with a slight overpressure of 15N₂ gas (&gt;98 atom% 15N; Cambridge Isotope), which was then allowed to equilibrate with continued vigorous stirring for at least 60 minutes. Incubations were initiated by mixing 2L of mesh-prefiltered mixed-later seawater (sampled from a hydrocast subsequent to that used for the 15N₂-enriched water) in a 1:1 ratio with the 0.2µm-prefiltered 15N₂-enriched water in 4L polycarbonate bottles. Parallel 14N₂-incubations were prepared using 0.2µm-prefiltered, degassed seawater equilibrated with N₂ gas of natural isotopic abundance (0.37 atom% 15N). Bottles were then incubated in on-deck incubators that were temperature controlled with surface seawater from the ship's flow-through system and shaded to mimic the light field at 25m depth.</p>
<p>Samples for proteomic analysis were collected by filtering the contents of the 4L incubation bottles onto 0.2µm-pore PES membrane filters (MilliporeExpress) under gentle suction. Filters were frozen at -80 degrees Celsius (ºC) shipboard immediately upon collection and transported to UChicago in a LN₂-cooled dry shipper for analysis. Proteins were extracted from filters by agitation in a Beadbeater (40 seconds), sonication (QSonica Q500, 10 minutes in 10-second on/off pulses, 85% amplitude), and heating (95ºC, 25 minutes), all in a reducing and denaturing buffer (1% LDS, 200 millimolar (mM) Tris pH 8.0, 10 mM DTT) followed by alkylation of cysteine thiols with 40 mM iodoacetamide. Proteins were then precipitated in 4 volumes of acetone in glass centrifuge tubes overnight at -20ºC and pelleted by centrifugation for 60 minutes at 7000 x g. Protein pellets were dried, redissolved in denaturing buffer (8M urea, 0.2% deoxycholate, 1M ammonium bicarbonate pH 8), and purified using a modified eFASP (enhanced Filter-Aided Sample Preparation) protocol (Erde, et al. 2014) in passivated Sartorius Vivacon 500 concentrators (30 kDa nominal cutoff). Purified proteins were digested on-filter with MS-grade trypsin (37ºC, overnight, 2 micrograms (µg)). Peptides were eluted from the concentrators and dried by vacuum centrifugation.</p>
<p>For LC-MS analysis, peptide samples were reconstituted in 2% acetonitrile + 0.1% formic acid. All samples were separated on a monolithic capillary C18 column (GL Sciences Monocap Ultra, 100µm ID x 200-centimeter (cm) length) using a water-acetonitrile + 0.1% formic acid gradient (2-50% AcN over 180 minutes) at 360 nanoliters per minute (nL/min) using a Dionex Ultimate 3000 LC system with nanoelectrospray ionization (Proxeon Nanospray Flex source). Mass spectra were collected on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) operating in a data-dependent acquisition mode, with one high-resolution (120,000 m/∆m) MS1 parent ion full scan triggering 15 Rapid mode MS2 CID fragment ion scans of selected precursors.</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-2050685 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=2050685
completed
Jacob Waldbauer
University of Chicago
773-702-8322
5734 S Ellis Ave
Chicago
Illinois
60637
USA
jwal@uchicago.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
Eppendorf 5430, 5424
Sartorius Vivacon 500 concentrators
Dionex Ultimate 3000 LC system
Orbitrap Elite mass spectrometer (Thermo Fisher Scientific)
12L Niskin bottles
QSonica Q500
theme
None, User defined
Centrifuge
Concentrator Device
High-Performance Liquid Chromatograph
Mass Spectrometer
Niskin bottle
ultrasonic cell disrupter (sonicator)
instrument
BCO-DMO Standard Instruments
KM2204
service
Deployment Activity
Hawaii Ocean Time-series Station ALOHA
place
Locations
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.
EAGER: Tracking marine diazotrophy with isotope-labeling proteomics
https://osprey.bco-dmo.org/project/869416
EAGER: Tracking marine diazotrophy with isotope-labeling proteomics
<p>Throughout much of the global ocean, nitrogen fixation – the conversion of abundant atmospheric nitrogen gas into chemical forms usable by life – is a crucial source of nutrients for biological activity. In particular, life needs nitrogen to make proteins, the essential biochemical machines that power all cells. Nitrogen fixation in the oceans is carried out by a variety of microorganisms, whose surprising diversity and ecology oceanographers are working to uncover. This project uses a novel mass spectrometry technique to directly track the flow of nitrogen from nitrogen gas, via nitrogen fixation, into proteins. These measurements reveal which nitrogen-fixing organisms are active in a given part of the ocean, what proteins they make with the nitrogen they fix, and how that nitrogen ultimately flows to feed the entire marine biological community. This information will better inform models and predictions of how marine microbial ecosystems, and the essential biogeochemistry they perform, responds to changing conditions in the ocean. This project engages an undergraduate student researcher, recruited from groups underrepresented in science, in both the field and laboratory work.</p>
<p>Diazotrophy (biological N2 fixation) is the largest input of fixed nitrogen to the ocean and is carried out by a wide diversity of marine microbes, but we have limited ability to quantify the N2-fixation activity of the full diversity of marine diazotrophs, or to resolve how the N they fix ultimately flows to supply the broader microbial community. Since the primary biosynthetic fate of fixed N2 is protein production, proteomics is poised to make substantial contributions to our understanding of the marine nitrogen cycle, and to the molecular physiology and ecological roles of diazotrophs in particular. This project brings a novel 15N-tracking proteomics methodology to bear on three outstanding questions in marine diazotroph ecology:</p>
<p>1) How is whole-community N2 fixation activity apportioned among different diazotroph taxa?<br />
2) How do diazotrophs and their symbiotic partners make biosynthetic use of the N they fix?<br />
3) How much diazotroph-derived N is redistributed to particular non-diazotroph taxa?</p>
<p>The investigators are conducting 15N2-tracking proteomics experiments at the Hawaii Ocean Time-series (HOT) Station ALOHA, assaying N incorporation from 15N-labeled dinitrogen into proteins of both diazotrophs and the broader microbial community. This is the first use of proteomics to track diazotrophic 15N2 incorporation, representing a novel, molecular-level approach for investigating marine nitrogen fixation that encompasses the entire microbial community with high taxonomic resolution. 15N-tracking proteomics data provides unique insight into the flow of nitrogen currency through its key biological accounts in cellular proteomes, and into the ecophysiology and biogeochemical roles of marine diazotrophs.</p>
<p>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.</p>
15N2Fix Proteomics
largerWorkCitation
project
eng; USA
oceans
Hawaii Ocean Time-series Station ALOHA
-158
-158
22.75
22.75
2022-03-26
2022-03-30
Station ALOHA
0
BCO-DMO catalogue of parameters from Metaproteomics analyses of mixed-layer water from Hawaii Ocean Time-series Station ALOHA, collected on R/V Kilo Moana cruise KM2204 in March 2022, and incubated with 15N2 to track biosynthetic incorporation by diazotrophic microbes
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
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
https://osprey.bco-dmo.org/dataset/958552/data/download
download
onLine
dataset
<p>Mixed-layer water (25 meters (m) depth) was sampled at Station ALOHA using 12-liter (L) Niskin bottles on the HOT CTD rosette. To prepare 15N₂-enriched seawater for incubations, water was filtered first through nitex mesh (250-micrometers (µm)) and then through 0.2 µm-pore filters (Millipak) into pre-cleaned 2L pyrex glass bottles with gas-port caps (Chemglass). Water was then degassed under vacuum with vigorous stirring (Velp MST magnetic stir plates) for at least 30 minutes. The evacuated headspace was then filled with a slight overpressure of 15N₂ gas (&gt;98 atom% 15N; Cambridge Isotope), which was then allowed to equilibrate with continued vigorous stirring for at least 60 minutes. Incubations were initiated by mixing 2L of mesh-prefiltered mixed-later seawater (sampled from a hydrocast subsequent to that used for the 15N₂-enriched water) in a 1:1 ratio with the 0.2µm-prefiltered 15N₂-enriched water in 4L polycarbonate bottles. Parallel 14N₂-incubations were prepared using 0.2µm-prefiltered, degassed seawater equilibrated with N₂ gas of natural isotopic abundance (0.37 atom% 15N). Bottles were then incubated in on-deck incubators that were temperature controlled with surface seawater from the ship's flow-through system and shaded to mimic the light field at 25m depth.</p>
<p>Samples for proteomic analysis were collected by filtering the contents of the 4L incubation bottles onto 0.2µm-pore PES membrane filters (MilliporeExpress) under gentle suction. Filters were frozen at -80 degrees Celsius (ºC) shipboard immediately upon collection and transported to UChicago in a LN₂-cooled dry shipper for analysis. Proteins were extracted from filters by agitation in a Beadbeater (40 seconds), sonication (QSonica Q500, 10 minutes in 10-second on/off pulses, 85% amplitude), and heating (95ºC, 25 minutes), all in a reducing and denaturing buffer (1% LDS, 200 millimolar (mM) Tris pH 8.0, 10 mM DTT) followed by alkylation of cysteine thiols with 40 mM iodoacetamide. Proteins were then precipitated in 4 volumes of acetone in glass centrifuge tubes overnight at -20ºC and pelleted by centrifugation for 60 minutes at 7000 x g. Protein pellets were dried, redissolved in denaturing buffer (8M urea, 0.2% deoxycholate, 1M ammonium bicarbonate pH 8), and purified using a modified eFASP (enhanced Filter-Aided Sample Preparation) protocol (Erde, et al. 2014) in passivated Sartorius Vivacon 500 concentrators (30 kDa nominal cutoff). Purified proteins were digested on-filter with MS-grade trypsin (37ºC, overnight, 2 micrograms (µg)). Peptides were eluted from the concentrators and dried by vacuum centrifugation.</p>
<p>For LC-MS analysis, peptide samples were reconstituted in 2% acetonitrile + 0.1% formic acid. All samples were separated on a monolithic capillary C18 column (GL Sciences Monocap Ultra, 100µm ID x 200-centimeter (cm) length) using a water-acetonitrile + 0.1% formic acid gradient (2-50% AcN over 180 minutes) at 360 nanoliters per minute (nL/min) using a Dionex Ultimate 3000 LC system with nanoelectrospray ionization (Proxeon Nanospray Flex source). Mass spectra were collected on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) operating in a data-dependent acquisition mode, with one high-resolution (120,000 m/∆m) MS1 parent ion full scan triggering 15 Rapid mode MS2 CID fragment ion scans of selected precursors.</p>
Specified by the Principal Investigator(s)
<p>Peptide sequences were identified from mass spectral data using Sequest HT implemented in Proteome Discoverer v2.2 (Thermo Scientific). diDO-IPTL quantitation (from 14N₂ incubations) was performed using MorpheusFromAnotherPlace (MFAP; Waldbauer et al., 2017; <a href="https://github.com/waldbauerlab" target="_blank">https://github.com/waldbauerlab</a>). Precursor and product ion mass tolerances for MFAP searches were set to 20ppm and 0.6Da, respectively. Static cysteine carbamidomethylation and variable methionine oxidation, N-terminal (d4)-dimethylation, and C-terminal 18O₂ were included as modifications. False discovery rate for peptide-spectrum matches was controlled by target-decoy searching to &lt;1%. Protein-level relative abundances and standard errors were calculated in R using the Arm postprocessing scripts for diDO-IPTL data (<a href="https://github.com/waldbauerlab" target="_blank">https://github.com/waldbauerlab</a>). Peptide atom %15N values (from 14N₂ incubations) were determined using the cPIE (classified peptide isotope enrichment) pipeline (Zimmerman, et al. 2023; <a href="https://github.com/waldbauerlab" target="_blank">https://github.com/waldbauerlab</a>).</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
Eppendorf 5430, 5424
Eppendorf 5430, 5424
PI Supplied Instrument Name: Eppendorf 5430, 5424 Instrument Name: Centrifuge Instrument Short Name: Instrument Description: A machine with a rapidly rotating container that applies centrifugal force to its contents, typically to separate fluids of different densities (e.g., cream from milk) or liquids from solids.
Sartorius Vivacon 500 concentrators
Sartorius Vivacon 500 concentrators
PI Supplied Instrument Name: Sartorius Vivacon 500 concentrators Instrument Name: Concentrator Device Instrument Short Name: Instrument Description: A concentrator is a device designed to increase the weight per unit volume of a substance.
This category includes vacuum centrifuge concentrator, which include a vacuum chamber within which a centrifuge rotor is mounted for spinning a plurality of vials containing a solution at high speed while subjecting the solution to a vacuum condition for concentration and evaporation. Alternative names: sample concentrator; speed vacuum; speed vac.
Dionex Ultimate 3000 LC system
Dionex Ultimate 3000 LC system
PI Supplied Instrument Name: Dionex Ultimate 3000 LC system PI Supplied Instrument Description:Dionex UltiMate 3000 RSLCnano w/ GL Sciences Monocap Ultra monolithic capillary C18 column with nanoelectrospray ionization (Proxeon Nanospray Flex source) Instrument Name: High-Performance Liquid Chromatograph Instrument Short Name:HPLC Instrument Description: A High-performance liquid chromatograph (HPLC) is a type of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of the mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by high pressure pumping of the sample mixture onto a column packed with microspheres coated with the stationary phase. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB11/
Orbitrap Elite mass spectrometer (Thermo Fisher Scientific)
Orbitrap Elite mass spectrometer (Thermo Fisher Scientific)
PI Supplied Instrument Name: Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) 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/
12L Niskin bottles
12L Niskin bottles
PI Supplied Instrument Name: 12L Niskin bottles Instrument Name: Niskin bottle Instrument Short Name:Niskin bottle Instrument Description: A Niskin bottle (a next generation water sampler based on the Nansen bottle) is a cylindrical, non-metallic water collection device with stoppers at both ends. The bottles can be attached individually on a hydrowire or deployed in 12, 24, or 36 bottle Rosette systems mounted on a frame and combined with a CTD. Niskin bottles are used to collect discrete water samples for a range of measurements including pigments, nutrients, plankton, etc. Community Standard Description: http://vocab.nerc.ac.uk/collection/L22/current/TOOL0412/
QSonica Q500
QSonica Q500
PI Supplied Instrument Name: QSonica Q500 Instrument Name: ultrasonic cell disrupter (sonicator) Instrument Short Name: Instrument Description: Instrument that applies sound energy to agitate particles in a sample.
Cruise: KM2204
KM2204
R/V Kilo Moana
Community Standard Description
International Council for the Exploration of the Sea
R/V Kilo Moana
vessel
KM2204
Tully Rohrer
University of Hawaiʻi at Mānoa
R/V Kilo Moana
Community Standard Description
International Council for the Exploration of the Sea
R/V Kilo Moana
vessel