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Molecular & Cellular Proteomics 4:1107-1121, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry Reveals Stimulation-dependent Multisite Phosphorylation^*,S

Elisabetta Boeri Erba^,,¶, Elena Bergatto, Sara Cabodi, Lorenzo Silengo, Guido Tarone, Paola Defilippi^,|| and Ole N. Jensen^,**

From the Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark and the Department of Genetics, Biology and Biochemistry, Medical School, University of Torino, Via Santena, 5/bis 10126 Torino, Italy

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation 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 residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser⁹⁶⁷) and revealed constitutive phosphorylation of Thr⁶⁶⁹, Ser⁹⁶⁷, Ser¹⁰⁰², and Tyr¹⁰⁴⁵ and stimulation-dependent differential phosphorylation of Tyr¹⁰⁶⁸, Tyr¹⁰⁸⁶, Ser¹¹⁴², Tyr¹¹⁴⁸, and Tyr¹¹⁷³. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8–3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide ¹¹³⁷GSHQISLDNPDYQQDFFPK¹¹⁵⁵; however, only Tyr¹¹⁴⁸ was phosphorylated upon EGF treatment; in contrast Ser¹¹⁴² was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.

^** A Lundbeck Foundation research professor. To whom correspondence may be addressed. Tel.: 45-6550-2368; Fax: 45-6550-2467; E-mail: jenseno{at}bmb.sdu.dk

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