Computational protocol: Potential molecular mechanisms of overgrazing-induced dwarfism in sheepgrass (Leymus chinensis) analyzed using proteomic data

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[…] Raw proteomic data were converted into mzXML data using ReAdW software (http://tools.proteomecenter.org/wiki/index.php?title=Software:ReAdW). mzXML data was then matched with the Swiss-Prot/TrEMBL database (release-2015_07) (http://www.uniprot.org/uniprot) [, ] using the PeptideProphet tool in Trans-Proteomic Pipeline software (http://www.proteomecenter.org) []. The matching results were obtain using pepXMLTab software. The following search parameter settings were used: peptide tolerance, ± 20 ppm and tandem mass spectrometry tolerance, 0.1 Da. A peptide was required to have at least a single assigned fragment and only unique peptides were used for protein identification. Protein with a false discovery rate (FDR) < 0.01 were considered to have high credibility. [...] Functional annotation was performed for the proteins with high credibility based on the Gene Ontology (GO) database (http://www.geneontology.org/) [] and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database (http://www.kegg.jp/kegg/pathway.html) [] using the enrichment analysis tool Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.8 []. The subcellular localisation of the proteins was determined through Cell-PLoc 2.0, a package of web servers for predicting the subcellular localisation of proteins in various organisms []. [...] The t-test method in limma package (http://www.bioconductor.org/packages/release/bioc/html/limma.html) was utilised to identify DEPs between GZ and NG groups. The p-value for each protein was adjusted by the Benjamini–Hochberg method []. Only proteins meeting the cut-off criteria of fold change > 1.2 and adjusted p-value < 0.05 were identified as DEPs. The DEPs with similar expression patterns were clustered with pheatmap software [].To reveal potential functions of DEPs, they were subjected to functional enrichment analysis based on the GO database, and only GO terms with p-value < 0.05 were considered significant. The pathway location of DEPs was determined using KEGG Mappertool (http://www.kegg.jp/kegg/mapper.html). […]

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