Computational protocol: Pepino mosaic virus Infection of Tomato Affects Allergen Expression, but Not the Allergenic Potential of Fruits

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Protocol publication

[…] Immunoblot analyses were carried out with protein extracts from ten pooled infected or non-infected fruits in three technical replicates. Two dimensional electrophoresis (2DE) was conducted following the protocol by Klose and Kobalz . Isoelectric focussing (first dimension) was performed in vertical rod gels containing 9 M urea, 4% acrylamide, 0.3% piperazine diacrylamide, 5% glycerine, 2% carrier ampholyte (pH 2-11), 0.06% TEMED, 0.08% ammonium persulfate. 60 µg of protein extract was focussed at 1,841 V. SDS–PAGE (second dimension) was performed in gels (0.1 cm×7 cm×8 cm; 15% acrylamide, 0.2% bisacrylamide, 375 mM Tris-HCl (pH 8.8), 0.1% SDS, 0.03% TEMED, and 0.08% ammonium persulfate). 2DE separations were performed in duplicate. One gel was stained with FireSilver (Proteome Factory, Berlin, Germany) for preparative applications; the other gel was used for immunoblotting. 2DE gels were blotted using an Immobilon-P membrane (PVDF, pore size 0.45 mm; Millipore, Bedford, USA) and a Trans-Blot SD Semi-Dry Transfer Cell (Biorad, Munich, Germany) at a constant current and 5 V over night at 4°C.After washing and blocking, membranes were incubated with a serum pool of nine tomato allergic subjects (diluted 1∶10 in TBS Tween containing 1% (w/v) BSA) over night and then incubated with peroxidase conjugated goat anti-human IgE (Sigma, Taufkirchen, Germany, diluted 1∶3,000 in TBS Tween containing 1% (w/v) BSA) for 2 h. Immunoblots were developed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, USA). Between all incubation steps the membrane was washed with TBS Tween (5 times for 10 min). Proteins were identified by Proteome Factory AG (Berlin, Germany). After in-gel digestion with trypsin, peptides were analysed in a nanoLC-ESI-MS/MS. Proteins were identified using MS/MS ion search of the Mascot search engine (Matrix Science, London, England) and a protein database (National Center for Biotechnology Information (NCBI), Bethesda, USA). MASCOT expresses the probability that peptides match at random to a given protein by a probability score. A score larger than 57 indicates identity or extensive homology (p = 0.05).Separate 2DE protein gels (20×30 cm) were used to quantify representative putative tomato allergens at the protein level. Identification and quantification was achieved by comparison with immunoblots. The 2DE gels were digitised at a resolution of 150 dpi using a PowerLook 2100XL scanner with transparency adapter. 2D image analysis and protein spot quantification was performed using the Proteomweaver software 3.1 (Definiens AG, Munich, Germany). [...] Statistical analyses were carried out using Statistica (version 9, Tulsa, OK, USA). RNA accumulation analyses were subjected to one-way analysis of variance (ANOVA) procedures (p = 0.05). The skin prick test and basophil test data were subjected to the non-parametric test. Medians were separated by the Mann-Whitney U test procedure (p = 0.05). […]

Pipeline specifications

Software tools Mascot Server, Proteomweaver, Statistica
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Solanum lycopersicum, Pepino mosaic virus
Diseases Drug Hypersensitivity, HIV Infections