Computational protocol: Crosstalk between proteins expression and lysine acetylation in response to patulin stress in Rhodotorula mucilaginosa

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Protocol publication

[…] The resulting MS/MS data was processed using MaxQuant with integrated Andromeda search engine (v.1.4.1.2). Tandem mass spectra were searched against protein database of Rhodotorula toruloides/Rhodosporidium toruloides, which were highly homologous with R. mucilaginosa and both are belonged to Rhodosporidium clade. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages, 5 modifications per peptide and 5 charges. Mass error was set to 10 ppm for precursor ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, Acethylation on Lysine, and acetylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for protein, peptide and modification site were specified at 1%. Minimum peptide length was set at 7. For quantification, TMT-6-plex was selected. All the other parameters in MaxQuant were set to default values. The site localization probability was set as >0.75. All the peptide sequences was deposited in the public protein peptide database MS-Viewer: http://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer. [...] Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (www. http://www.ebi.ac.uk/GOA/). Firstly, Converting identified protein ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt-GOA database, the InterProScan soft would be used to annotated protein’s GO functional based on protein sequence alignment method by Blast2GO software. Then proteins were classified by Gene Ontology annotation based on biological process, molecular function and cellular component. Domain functional description was annotated by InterProScan based on protein sequence alignment method, and the InterPro domain database (http://www.ebi.ac.uk/interpro/) was used. We used Wolf PSORT to predict subcellular localization. Soft motif-x was used to analysis the sequences constituted with amino acids in specific positions of modify-21-mers (10 amino acids upstream and downstream of the site) in all protein sequences. And all the database protein sequences were used as background database parameter, other parameters with default.To generate the PPI network, proteins that were selected from the all identified proteins were searched against the STRING database (v10.0) for protein-protein interactions. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. Interactions that have a STRING high confidence score (score ≥ 0.7, without “text-mining evidence”, see STRING instructions for details) were fetched. CytoScape (v3.3.0) was applied to visualize the interaction network generated from STRING. […]

Pipeline specifications

Software tools MaxQuant, MS-Viewer, InterProScan, Blast2GO, WoLF PSORT
Databases UniProt-GOA
Applications MS-based untargeted proteomics, Protein sequence analysis, Amino acid sequence alignment
Organisms Rothia mucilaginosa
Chemicals Zinc