Computational protocol: A phloem-limited fijivirus induces the formation of neoplastic phloem tissues that house virus multiplication in the host plant

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Protocol publication

[…] Each sample was pooled from six plants at the same pre-flowering stage. Protein samples were prepared as described previously. Protein concentration was quantitative using a 2-D Quant Kit (GE Healthcare, USA) according to the manufacturer’s instructions and the amount of loaded protein samples was calculated. The resulting protein samples were then subjected to electrophoretic analysis using an optimised procedure for DIGE (GE Healthcare, UK) according to the manufacturer’s instructions. Prior to electrophoresis, protein samples were labelled using the CyDyes DIGE fluors (Cy3 and Cy5) (GE Healthcare). After electrophoresis, the 2-DE gels were visualised using an Ettan DIGE Imager (GE Healthcare). The gel images were transferred to the DeCyder 2D 7.0 software package (GE Healthcare) and further analysed with Differential In-gel Analysis modules (GE Healthcare). Protein level differences between two samples were analysed with the DeCyder-BVA (Biological Variation Analysis 5.02) module. MALDI-TOF-MS was performed by Shanghai Applied Protein Technology Co., Ltd. Briefly, silver stained protein spots were excised from the 2-DE gel and treated. The samples were then loaded on the MALDI target together with CHCA as a matrix for MALDI-TOF-MS analysis. All procedures were performed as described previously. ProFound software was further used to identify protein spots from the NCBI non-redundant database via PMF experiments. All candidates with more than three identified CID spectra of peptides targeting the same protein and SEQUEST Xcorr values greater than 2.0 were further assessed by the comparison with Mr and Pi experimental values obtained from 2-DE. The sequences of identified proteins were further subjected to BLASTp to identify their homologous proteins and Blast2GO for functional annotation analysis. […]

Pipeline specifications

Software tools Comet, BLASTP, Blast2GO
Application MS-based untargeted proteomics
Organisms Viruses, Human poliovirus 1 Mahoney, Rice black streaked dwarf virus, Zea mays
Diseases Neoplasms, HIV Infections, Mitochondrial Diseases
Chemicals Amino Acids