Computational protocol: Mitogen-activated protein kinase signaling causes malignant melanoma cells to differentially alter extracellular matrix biosynthesis to promote cell survival

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Protocol publication

[…] Tandem mass spectra were searched against a concatenated target-decoy database containing the forward and reverse sequences of the target database using the Proteome Discoverer 1.4 and SEQUEST search engine (Thermo Finnigan). The target sequence was downloaded from a human Uniprot database (database released on November 13, 2013) and 124 common contaminant proteins. The search algorithm was configured to specify the following parameters: precursor tolerance, 10 ppm; fragment tolerance, 0.5 Da; static modification, cysteine carbamidomethylation; and fully tryptic status. SEQUEST search results from Proteome Discoverer were further analyzed by Scaffold (Proteome Software Inc., Portland, OR). The discriminant score was set such that a false positive rate of 1 % was determined based on the number of accepted decoy database peptides. Scaffold outputs label-free quantification based on precursor ion intensity for data from Proteome Discoverer. Precursor intensity refers to the area under an MS1 spectrum peak corresponding to a specific peptide. We used normalized total precursor intensity to derive quantitative values in this paper. Statistical analysis was performed by Qlucore Omics Explorer (Qlucore AB, Lund, Sweden). Two group comparisons (a paired t-test) was used to compare the mean value of relative abundance based on normalized total precursor intensity from three biological replicates for each protein identified in conditioned media collected in day 2 and day 10. The heatmap was generated based on hierarchical clustering of global protein expression filtered by the p-value (p < 0.05). […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Melanoma, Neoplasms