Computational protocol: Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition

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Protocol publication

[…] pTyr peptides were enriched from tryptic digests of cell lysates as described () except that lysates were subjected to brief PAGE and in-gel trypsin digestion. LC-MS-MS analysis of immunoaffinity-purified peptides was performed as described () with modifications described in . MS/MS peptide spectra were acquired using data-dependent scanning in which one full MS spectrum was followed by 5 MS/MS spectra. A data-dependent scan for the neutral loss of phosphoric acid or phosphate resulted in acquisition of an MS/MS/MS of the neutral loss ion. Proteins were identified from mass spectra using the Myrimatch algorithm and the human IPI database (v. 3.56) (). Data were filtered using a 2% FDR for all peptides using the IDPicker algorithm () allowing for a single peptide/spectrum match. […]

Pipeline specifications

Software tools MyriMatch, IDPicker
Application MS-based untargeted proteomics
Organisms Homo sapiens, Mus musculus
Diseases Breast Neoplasms, Neoplasms
Chemicals Potassium