Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamics.
Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein-protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N-terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling.
Schuchardt
Sven
S
Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine ITEM, Nikolai-Fuchs-Strasse 1, Hannover, Germany.
Borlak
Jürgen
J
D000818:Animals; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D004815:Epidermal Growth Factor; D020794:Receptor Protein-Tyrosine Kinases