Computational protocol: SILAC-Based Quantitative Proteomic Analysis of Diffuse Large B-Cell Lymphoma Patients

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Protocol publication

[…] Raw data, containing full-scan spectra acquired in profile mode and centroid MS/MS spectra, from the analysis of tryptic peptides, were merged and processed using the MaxQuant software version 1.2.0.18 []. Default settings were used for feature extraction and first search for recalibration was performed against the human first search database provided with the software. The Andromeda search engine [] integrated into the MaxQuant package was used for peptide identification and searches were performed against the human subsection of the UniProtKB database. Parameters for identification and quantification were set as follows: variable modification: oxidation of methionine and acetylation of the N-terminal; fixed modification: carbamidomethylation of cysteine, MS/MS tolerance 0.5 Da; peptide and protein false discovery rate (FDR) was set to 0.01 and for SILAC labeled samples the heavy label was set to arg6 and lys6. SILAC protein ratios are determined as the median of all peptide ratios assigned to the protein. For quantification a minimum peptide ratio count of two was set for each protein. To ensure that the Log2 values of the normalized protein H/L ratios followed a normal distribution and were centered to zero; histograms were plotted. A two sample t-test was performed to determine significant differences in protein ratios between the groups, Perseus module (version 1.2.0.17) available in the MaxQuant environment. [...] The differentially expressed proteins in our study were analyzed using the DAVID (Database for Annotation, Visualization and Integrated Discovery) Bioinformatic resources version 6.7 (http://david.abcc.ncifcrf.gov), the PANTHER (Protein ANalysis THrough Evolutionary Relationships) system version 7 (http://www.pantherdb.org), and the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) version 9.1 (http://string.embl.de/). The list of Uniprot Accession IDs was loaded into the online tool to extract and summarize functional classification. In DAVID, to identify proteins in the most important biological functional groups, the Functional Annotation Clustering tool was used with an EASE threshold of 0.01. Also, to rank the overall importance of proteins, DAVID uses the term “enrichment score”; a higher score for a group indicates that the proteins are involved in more important terms (enrichment score of 1.3 is equivalent to nonlog scale 0.05). PANTHER performs a classification of genes and proteins and provides gene ontology terms, biological processes, molecular function, and pathways. We also used the STRING database for physical and functional interactions among the identified proteins. STRING uses a score to define interaction confidence; all interactions with a confidence score >0.7 (high confidence) were collected. […]

Pipeline specifications

Software tools MaxQuant, Andromeda, Perseus, DAVID, PANTHER
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Homo sapiens
Diseases Lymphoma, Neoplasms, Lymphoma, B-Cell