Identification ubiquitination of EGFR by LC-MS/MS

Previously, analysis of EGF-induced EGFR ubiquitination by mass spectrometry revealed that ubiquitinated lysine residues were located in the TKD of EGFR. However, the specific lysine residues for the CBL-mediated polyubiquitination have not been identified. To investigate the differences in EGFR ubiquitination by ZNRF1 and CBL, we sought to identify the acceptor residues for polyubiquitin chains mediated by these two E3 ubiquitin ligases. We co-transfected HEK293T cells with EGFR and ZNRF1 or CBL, and immunoprecipitated EGFR for liquid chromatography-tandem mass spectrometry analysis.