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PSM1250: knockdown collected 72h after the last feeding with dsRNA sample 4 Parent Study: PSE129 Organism: Apis mellifera Description Larva, collected 72h after the last feeding with dsRNA Statistics Proteins: 326 Peptides: 638 Spectra: 1406 Dates Release: June 23, 2010 Deposit: January 8, 2010 Metadata update: December 20, 2010 Data update: December 20, 2010 Contact Organization: Norwegian University of Life Sciences / Arizona State University Address: PO Box 5003 Aas N-1432 Preparation Growth: In vitro until sampled. Treatment: Application of gfp dsRNA or irs dsRNA. Larvae were fed dsRNA. Extract: Proteins were extracted by grinding and boiling samples in a buffer containing SDS and beta-mercaptoethanol. Contaminants/undesired molecules were removed by methanol/chloroform precipitation. Digestion: 40 µg per sample were subjected to digestion over night at 30°C with 1 µg of trypsin (sequencing grade, Roche, Indianapolis, IN, USA) in digestion buffer (50 mM tris pH 8.5, 0.15 M NaCl, 1 mM CaCl2). Separation: Peptides were separated via C18 HPLC before MS analysis. Characteristics Developmental stage: Larva Instrumentation Platform: ESI-ION-TRAP Manufacturer: Thermo Electron, San Jose, CA Ion source: Nano electrospray Analyzer: Ion trap Detector: EMT Setup: Linear ion trap mass spectrometer equipped with a nano electrospray interface operated in the positive ion mode. Typical instrument settings included a spray voltage of 1.5 kV and an ion transfer tube temperature of 200°C. Voltages across the capillary and the quadrupole lenses were tuned for optimal signal intensity using the +2 ion of angiotensin I (m/z 649). The scan sequence consisted of 1 full MS scan followed by 5 MS/MS scans of the five most abundant ions. Ions were dynamically excluded over a period of 40 sec, with a maximum of 500 ions excluded. Repeat count was set to 2. Processing Methods Software: OMSSA Parameters: Using the open source search tool OMSSA (version 2.0.0) the spectra were matched against an A. mellifera sequence database retrieved from NCBI (http://www.ncbi.nlm.nih.gov/) containing additional trypsin and keratin sequences. The following filtering criteria were used: 0.8 Da fragment tolerance, 0.8 Da precursor tolerance, maximum of 2 missed cleavages, only tryptic sequences allowed, initially ten possible peptide hits per spectrum reported then filtered to one peptide hit per spectrum, variable modifications: methionine oxidation, deamidation of N and Q. Acceptance threshold for peptides: e = 0.1. At least two peptides per protein were required for protein identification. Quantification: Quantification for proteins required the presence of a spectral count = 3 in 3 or more of the 4 replicates of one group and the identification of at least 2 unique peptides in at least 3 out of 4 replicates in one group. Missing spectral count values were replaced by 0.1. Individual spectral counts were divided by the total spectral count to account for possible variations in total protein amount. Further statistical analysis was conducted as described previously (PMID: 17805521). Search Engines: OMSSA Modifications Variable: Deamidated (7), Oxidation (35) |