Computational protocol: Activation of diverse signaling pathways by oncogenic PIK3CA mutations

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Protocol publication

[…] Proteome Discoverer (v 1.3; Thermo Fisher Scientific) suite was used for quantitation and database searches. The tandem mass spectrometry data were searched using Mascot (Version 2.2.0) and SEQUEST search algorithms against a Human RefSeq database (v 46 containing 33,249 entries) supplemented with frequently observed contaminants. For both algorithms, the search parameters included a maximum of one missed cleavage; carbamidomethylation of cysteine as a fixed modification; N-terminal acetylation, oxidation at methionine, phosphorylation at serine, threonine and tyrosine and SILAC labeling 13C6,15N2-lysine; 2H4-lysine; 13C6-arginine and 13C6,15N2-arginine as variable modifications. The MS tolerance was set at 10 ppm and MS/MS tolerance to 0.1 Da. Score cut off was set to 0.01 false discovery rate at the peptide level. The probability of phosphorylation for each Ser/Thr/Tyr site on each peptide was calculated by the PhosphoRS algorithm . We averaged the intensities of phosphopeptides identified from the forward and reverse experimental groups. We chose a 1.5 fold cut off to consider peptides as phosphorylation increased and a 0.67 fold for peptides to be considered as phosphorylation decreased. This threshold was chosen because we observed that AKT phosphorylation increased by 1.53 fold in PIK3CA mutant knockin cells. Many of the known phosphorylation sites of AKT substrates were found to range between 1.5 and 2-fold in the knock-in cell lines compared to the parental cells. Among these were BAD S75 (1.82), CTNNB1 S552 (1.62), HSPB1 S82 (1.81), PDCD4 S457 (1.50), PEA15 S116 (1.60), RANBP3 S126 (1.96) and YAP1 S127 (1.65). To identify and plot the differentially regulated phosphopeptides, we averaged the intensity of each phosphopeptide detected from MCF10A, PIK3CA mutant knockin cells and knockin cells treated with PIK3CA inhibitor, J124. The relative ratio was calculated by dividing the intensity of each phosphorylated peptide over the average intensity of the corresponding peptide and used for plot. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository. […]

Pipeline specifications

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