Protein concentrations were measured using the BCA Protein Assay (Thermo
Fisher Scientific). Reduction, alkylation, and trypsin digestion were performed
as previously described (32 (link)). For
phosphotyrosine peptide enrichment, peptide immunoprecipitation was performed
using p-Tyr-100 phosphotyrosine antibody beads (Cell Signaling Technology) as
previously described (32 (link)). Prior to
immunoprecipitation, a 5 pmol fraction of synthetic phosphopeptide LIEDAEpYTAK
was added to each individual replicate and time point sample as a normalization
standard to correct for any variation in mass spectrometer sensitivity between
individual replicates. Samples were then desalted using ZipTip pipette tips (EMD
Millipore, Billerica, MA) according to the manufacturer’s
instructions.
Fisher Scientific). Reduction, alkylation, and trypsin digestion were performed
as previously described (32 (link)). For
phosphotyrosine peptide enrichment, peptide immunoprecipitation was performed
using p-Tyr-100 phosphotyrosine antibody beads (Cell Signaling Technology) as
previously described (32 (link)). Prior to
immunoprecipitation, a 5 pmol fraction of synthetic phosphopeptide LIEDAEpYTAK
was added to each individual replicate and time point sample as a normalization
standard to correct for any variation in mass spectrometer sensitivity between
individual replicates. Samples were then desalted using ZipTip pipette tips (EMD
Millipore, Billerica, MA) according to the manufacturer’s
instructions.
