Computational protocol: Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

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Protocol publication

[…] The peak list files containing MS and MS/MS data were analyzed using MaxQuant version 1.4.08[]. For protein identification, the MS/MS data was searched against the Uniprot protein database which includes the Swissprot (manually annotated and reviewed) and TrEMBL (automatically annotated and not reviewed) databases []. The database was searched using the MASCOT search engine utilizing Andromeda as the peptide search algorithm that is incorporated in the MaxQuant platform []. The search was conducted using the following settings: trypsin cleavage with a maximum of two missed cleavages, fixed modification of iodoethanol addition to cysteine, variable modification of oxidation of methionine. The MS mass tolerance was set at 4.5 ppm with a maximum number of five modifications. The false discovery rate was set at 0.01 for proteins and peptides and was run against a decoy revert database. Peptides required a minimum length of seven amino acids. The MS/MS tolerance was set at 0.1 Da for protein identification. The minimum score for modified and unmodified peptides was set at forty. At least two peptides were required for protein identification.The bioinformatics and statistical package Perseus 1.4.1.3 was used to analyze the MaxQuant output. Contaminants identified by Perseus, such as keratin, were removed from the analysis. For label free quantitation (LFQ), the intensity for each protein was transformed log2(x) and samples who had a peptide intensity below the level of detection were assigned a value of 14. We limited the analysis to proteins which were identified in at least 3 out of the 4 biological replicates.Proteins identified were clustered by their Gene Ontology (GO) Term based on biological process or molecular function using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v 6.7 [, ]. Visualization of protein interactions was accomplished using STRING version 9.1[]. We used the confidence view with a score of 0.4, indicating a medium confidence level. We did not limit the number of interactions in the analysis. […]

Pipeline specifications

Software tools MaxQuant, Mascot Server, Andromeda, Perseus, DAVID
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Pneumonia, Lipid
Chemicals Acyl Coenzyme A