Phosphotyrosine Peptide Identification and Quantification

After phosphotyrosine immunoprecipitation at the peptide level according to the manufacturer's instructions, tryptic phosphopeptides were eluted with 0.15% trifluoroacetic acid and concentrated to 20 μl using vacuum centrifugation (Speedvac, Thermo, San Jose, CA, USA). A nanoflow liquid chromatograph (U3000, Dionex, Sunnyvale, CA, USA) coupled to an electrospray ion trap mass spectrometer (LTQ-Orbitrap, Thermo) was used for tandem mass spectrometry peptide sequencing experiments for identification and relative quantification. Each sample was analyzed in duplicate, as previously described.^{52 (link)}