Computational protocol: Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins

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Protocol publication

[…] Data analysis was performed as described earlier [, ] with slight modifications. Briefly, data from LC-MS/MS analysis were analyzed using the open-source software framework OpenMS [] and the OpenMS Proteomic Pipeline (TOPPAS) []. For peptide and protein identification, mzML-files were searched against a human decoy-database (Swiss-Prot, www.uniprot.org, 20,161 entries) using two different search engines (open mass spectrometry search algorithm (OMSSA) [] XTANDEM []). The search was performed with a precursor mass tolerance of 35 ppm and a fragment mass tolerance of 0.1 Da. Carbamidomethylation on cystein residues was considered as a fixed modification. An oxidation of methionine residues as well as a 13C6-label on arginine and lysine residues were considered as variable modifications. Peptides were identified with a q-value of 0.05. For SILAC quantification the raw data files were converted to *.mzXML in profile mode using massWolf file converter. Further data processing was carried out with TOPPAS. The *.mzXML files were converted to *.mzML. For subsequent data processing the mzML files were filtered (only MS1 level, rt-range [s]: 600–2400) and smoothed (savitsky golay algorithm, frame length: 13, polynomial order: 4). SILAC pairs were detected and quantified using SILACAnalyzer with the following parameters: one missed cleavage, retention time threshold 10 s, intensity cutoff 20 counts, intensity correlation 0.7 and a model deviation of 1.8. A peptide required at least three isotopic peaks and maximal seven isotopic peaks to be taken into account by the SILACAnalyzer. Detected SILAC pairs were exported as *.consensusXML and matched with peptide identifications (*idXML) using IDMapper (retention time tolerance: 10 s, m/z-tolerance: 1 Da). Results were exported as *.csv and further statistical processing was carried out using mathematica. […]

Pipeline specifications

Software tools OpenMS, TOPPAS, OMSSA, massWolf
Databases UniProt
Application MS-based untargeted proteomics
Diseases Head and Neck Neoplasms, Neoplasms