Computational protocol: Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis

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Protocol publication

[…] We used Proteome Discoverer™ (PD) software version 1.3 (Thermo Scientific) to perform the quantitative proteomic analysis. The MS/MS spectra search was performed by SEQUEST® engines to search against the Uniprot Homo sapiens database (http://www.uniprot.org), coupled to the appropriate statistical and quantitative validation methods. Starting with the raw data, PD calculated the relative intensities of reporter ions from a specific identified tandem mass spectrum. A threshold intensity rate of 10,000 for the sum of the reporter ion intensities of HCD spectra was applied. A first statistical evaluation of these large data sets was performed by computing the distribution of m/z measurements. We evaluated analysis performance in terms of molecular weight (MW) and isoelectric point (pI) range of detected proteins. Data derived from MS analysis were examined using percolator for false discovery rate (FDR) < 0.01, which was calculated on the basis of the number of peptide matches against a decoy database. We then performed MS analysis of the TmT labeled samples on Q Exactive. Protein intensities resulted from the average of the single TmT reporter ion intensities obtained for each peptide associated with a specific protein. The average ratio of differential TmT 127/126 expression (1.5 fold increase or decrease) represents the ratio of two samples. In other words, we identified DEPs in our TmT experiment using 1.5 and 0.67 as the up-regulation and down-regulation cutoff points. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet, Percolator
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Breast Neoplasms
Chemicals Doxorubicin, NADP, Glucose-6-Phosphate