Computational protocol: Potato skin proteome is enriched with plant defence components

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Protocol publication

[…] Protein identification was performed at the Smoler Proteomics Center at the Technion, Israel. Briefly, protein spots were excised from the gel, treated with 100 mM iodoacetamide and trypsinized with modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. The resulting tryptic peptides were resolved by reverse-phase chromatography on 0.1×200 mm fused silica capillaries (100 μm ID, J&W, USA) packed with Everest reverse-phase material (Grace Vydac, USA). The peptides were eluted with linear 50 min gradients of 5–95% (v/v) acetonitrile with 0.1% (v/v) formic acid in water at a flow rate of 0.4 μl min−1. Mass spectrometry (MS) was performed in an ion-trap mass spectrometer (LCQ-DecaXP, Finnigan, USA) in positive mode using a repetitively full MS scan followed by collision-induced dissociation (CID) of the three most dominant ions selected from the first MS scan.The MS data were clustered and analysed using Sequest () or Pep-Miner () software. Briefly, the Sequest software identifies uninterpreted peptides with tandem mass spectra produced under low-energy (10–50 eV) collision conditions by correlating their mass-to-charge ratios for fragment ions to those predicted for known amino acid sequences obtained from the Genepept database. The Pep-Miner software manages large amounts of raw data obtained from the MS by clustering similar spectra from multiple runs, thereby reducing the amount of data to a manageable size and allowing fast and reliable identification of peptides in complex mixtures.Identifications were carried out using the NCBI non-redundant protein database from September 2006 and the TIGR potato EST database from February 2007. A comparison of the results obtained from the two databases is described in Table S1 of the Supplementary data at JXB online. […]

Pipeline specifications

Software tools Comet, PEP-Miner
Application MS-based untargeted proteomics
Organisms Solanum tuberosum