Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry

Multisite phosphorylation of proteins is a general mecha-nism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elu-cidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated res-idues of the human epidermal growth factor (EGF) recep-tor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimu-lation, i.e. by sodium pervanadate, EGF, and integrin-de-pendent adhesion. The contribution of cell-matrix adhe-sion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed a