URLhttps://www.bco-dmo.org/dataset/636756
Download URLhttps://www.bco-dmo.org/dataset/636756/data/download
Media Typetext/tab-separated-values
CreatedJanuary 28, 2016
ModifiedAugust 19, 2016
StateFinal no updates expected
Brief DescriptionRaw LC-MS/MS data, with list of identified peptides, and DNA sequences.

Acquisition Description

A proteomic profile of the chemolithoautotrophic, neutrophilic, iron-oxidizing Mariprofundus ferrooxydans was obtained in duplicates and analyzed via LC-MS/MS. Additionally, a proteomic analysis of the membrane fraction is included. Cultures were harvested at late log phase by following the protocol in Barco and Edwards (2014). Briefly, proteins were extracted with 0.1N NaOH/2% SDS solution, clarified and concentrated by nanofiltration. The concentrated samples were run on SDS-PAGE gels in duplicate lanes. All the bands from each lane were excised and analyzed via LC-MS/MS. Membrane fractions were obtained in the following way: the cell-separation protocol was performed per Barco and Edwards (2014) and the cell pellet was resuspended in 0.5M NaCl/40mM Tri-base buffer at pH 8.5 and sonicated for 10 cycles of 30 secs pulses followed by 30 secs of cooling. This sample was clarified and the supernatant was ultracentrifuged at 100,000 x g for 2 hrs. The reddish pellet was solubilized with 0.5% Ultrol Grade, n-Dodecyl-beta-D-maltoside and run on a SDS-PAGE gel. DNA: DNA was extracted by using the FastDNA Spin soil kit according to the manufacturer’s instructions (MP Biomedicals). Extracted DNAwas stored at 20 degrees C until processing. Genomic gaps were amplified by PCR on a Veriti thermal cycler (Life Technologies) as follows: 1 step of denaturation at 95 degrees C for 4 min; 35 cycles of denaturation, melting, and extension (95 degrees C for 30 s, 51 degrees C for 30 s, and 72 degrees C for 3 min, respectively); 1 step of extension at 72 degrees C for 10 min; and 1 final step of cooling at 4 degrees C. Primers used for the gap containing the Cyc1PV-1 gene were 79F (5'-GAAGCGATGGGAAATGTGAAT-3') and 375F (5'-CACACTGGAAGATGTTCTGG-3'). Primers used for the gap containing the Cyc2PV-1 gene were 555F (5'-ACTGATGGGTATCAACAACC-3') and 92R (5'-CCTATCTGTACCGAGCATTC-3'). The amplicons were purified by using the QIAquick PCR purification kit (Qiagen). Amplicon sizes were checked in a 1% agarose gel via electrophoresis. Purified PCR amplicons were submitted for Sanger sequencing and primer walking (Laragen).

Processing Description

Excised bands were trypsin-digested and submitted for LC-MS/MS analysis which involved a Thermo LTQ-Orbitrap XL mass spectrometer equipped with an Eksigent Nanoliquid Chromatography 1-D plus system. The resulting MS/MS spectra were searched against the proteomes of Mariprofundus ferrooxydans strains PV-1(Uniprot database) and M34 (IMG database) using Proteome Discoverer SEQUEST Daemon search engine. DNA: sequences were analyzed in Geneious v. R6 (Biomatters Ltd.).

Raw LC-MS/MS files are publicly available through the PRIDE Archive at http://www.ebi.ac.uk/pride/archive/projects/PXD001050 and http://www.ebi.ac.uk/pride/archive/projects/PXD001439.

DNA sequences were deposited in Genbank under accession numbers KR106296, KR106297, KR091570, and BK009249.

Instruments

General term for a laboratory apparatus commonly used for performing polymerase chain reaction (PCR). The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

(adapted from http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html)

Thermo LTQ-Orbitrap XL [Mass Spectrometer]
Details
Instance Description (Thermo LTQ-Orbitrap XL)

Excised bands were trypsin-digested and submitted for LC-MS/MS analysis which involved a Thermo LTQ-Orbitrap XL mass spectrometer equipped with an Eksigent Nanoliquid Chromatography 1-D plus system.

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.

Parameters

Details
type

Description of sample type.

text description of type of sample collected (rock, bio, fluid, etc)
taxon [taxon]
Details
taxon

Taxon studied.

taxonomic group or entity. This may be a family, class, genus, species, etc.; usually this parameter will contain a mixture of taxonomic entities.

repository [external_link]
Details
repository

Name of the repository where the raw data are stored.

Link to an external data entry.

identifier [accession number]
Details
identifier

Unique identifer: Either the PRIDE database project identifier or the NCBI accession number.

Database identifier assigned by repository and linked to GenBank or other repository.
Details
URL

Link to the PRIDE database or NCBI.

Link to an external data entry.

description [brief_desc]
Details
description

Description of the method and sample.

brief description, open ended, specific to the data set in which it appears

Dataset Maintainers

NameAffiliationContact
Roman A. BarcoBigelow Laboratory for Ocean Sciences
Katrina J. EdwardsUniversity of Southern California (USC)
Shannon RauchWoods Hole Oceanographic Institution (WHOI BCO-DMO)

BCO-DMO Project Info

Project TitleProteomic profiling of neutrophilic, iron-oxidizing Mariprofundus ferrooxydans, strain PV-1
AcronymProteomic profiling of Mariprofundus ferrooxydans
URLhttps://www.bco-dmo.org/project/632643
CreatedJanuary 13, 2016
ModifiedJanuary 13, 2016
Project Description

Description from C-DEBI website:
The aim of this proposal is to gain a better understanding of what subsets of proteins are actually being expressed during neutrophilic, microbial iron (Fe)-oxidation. The recently isolated Mariprofundus ferrooxydans, strain PV-1, will be used as a marine model organism to investigate proteomic differences under different Fe substrates: aqueous Fe2+ and solid Fe0. Two-dimensional gel electrophoresis (2D-GE) and shotgun proteomic methods (LC-MS/MS) will be employed to obtain results from the cultures grown under different conditions. The research being proposed would constitute the foundation for the development of diagnostic tools for the accordance, distribution, and activity level of Fe-oxidation, a globally important biogeochemical process at and below the ocean floor.

This project was funded by a C-DEBI Graduate Student Fellowship.

Project Maintainers
NameAffiliationRoleContact
Roman A. BarcoBigelow Laboratory for Ocean SciencesPrincipal Investigator
Katrina J. EdwardsUniversity of Southern California (USC)Co-Principal Investigator
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