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ABSTRACT:
PROVIDER: PRJNA1123917 | ENA |
REPOSITORIES: ENA
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Project description:These samples were collected from Stordalen Mire in Abisko Sweden on August 2010 from a core at 24-27cm. Microbial proteins were extracted from an in situ lysis protocol with 5% SDS and boiling. Peptides were analyzed by 22hr 2D-LC-MS/MS. A 22-hr 11-step two-dimensional separation was performed with a U3000 HPLC and ionized into the MS via nanospray ionization. During the full chromatographic runs, the MS oscillated between full scans in the Orbitrap at 30K resolution and 20 data-dependent MS/MS spectra in the dual linear ion trap. The entire run was controlled by Xcalibur, with dynamic exclusion on, and 2 microscans averaged for both the full scans and MS/MS scans . Three technical replicates were analyzed. Data analysis: The resultant MS/MS spectra were analyzed by an automated proteome informatics pipeline that uses SEQUEST and DTASelect. Raw spectra are extracted via readW program (ISB version 4.3.1), MS/MS spectra were searched against a database consisting of (i) metagenomic contigs from the same sample (consisting of 3,348,142 proteins predicted using Prodigal 2.50 (ii) wheat (NCBI February 2010, Protein sequences from NCBI taxonomy ID 4565) and (iii) with the common contaminants trypsin and human keratin. SEQUEST Searches using these databases were performed with the following parameters: Parent Mass Tolerance, 3.0; Fragment Ion Tolerance, 0.5; up to 4 missed cleavages allowed, and fully tryptic peptides only. No other modifications were analyzed. SEQUEST outputs were sorted and filtered via DTASelect with the following parameters: delta CN, >0.08; Xcorr, >1.8 (+1), 2.5 (+2), >3.5 (+3); +1 minimum charge state, +3 maximum charge state; and 2 peptides/protein.
2014-02-03 | PXD000410 | Pride
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