Computational protocol: A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis*

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Protocol publication

[…] Raw files of each double- and triple-labeling Jurkat T cell experiment were analyzed together using the in-house-built software MaxQuant (version 1.1.1.35 (, )). Each raw file from a particular slice was defined as a separate experiment in the experimental design file to obtain peptide ratios for each peptide in each slice. The derived peak list was searched with Andromeda () against the human International Protein Index protein sequence database (ipi.HUMAN.v3.68.fasta; 87,083 entries) supplemented with 262 frequently observed contaminants such as human keratins, bovine serum proteins, and proteases and concatenated with the reversed copies of all sequences. We required strict enzyme specificity with cleavage C-terminal after K, R, or D (trypsin + Asp-C), allowing up to two missed cleavage sites. Fixed modifications of cysteine carbamidomethylation (Cys 57.021464 Da) and variable modifications for the N-acetylation of proteins (N-term 42.010565 Da) and the oxidation of methionine (Met 15.994915 Da) were specified. Double or triple labeling was defined accordingly. The minimum peptide length was set as six amino acids. Scoring was performed in MaxQuant as described previously (). Parent masses and fragment ions were searched with initial mass tolerances of 7 ppm and 0.5 Da, respectively. False discovery rates (FDRs) at the peptide and protein levels were fixed at 1%, including automatic filtering on peptide length, mass error precision estimates, and peptide scores of all forward and reversed peptide identifications. The re-quantification feature was enabled. Reported protein groups had to be identified by at least one “razor peptide” (a peptide most likely belonging to the protein group) in order to be accepted. Quantitation was based on unique and razor peptides only, and a minimum of two ratio counts was required. Complete protein and peptide lists, as well as the underlying RAW files, are available in the Proteome Commons Tranche database. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Databases IPI
Application MS-based untargeted proteomics
Diseases Neoplasms