Schuchardt Sven, Borlak Jürgen
Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine ITEM, Nikolai-Fuchs-Strasse 1, Hannover, Germany.
Mass Spectrom Rev. 2008 Jan-Feb;27(1):51-65. doi: 10.1002/mas.20155.
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.
识别信号网络中的蛋白质已备受关注，因为一系列生物学过程完全依赖于蛋白质间的相互作用和蛋白质 - 蛋白质相互作用。蛋白质翻译后修饰（PTM）增加了另一层复杂性，从而形成复杂的信号网络。我们的研究小组特别感兴趣的是表皮生长因子（EGF）受体的信号网络，EGF受体是一种跨膜受体酪氨酸激酶，参与多种细胞过程，包括细胞增殖、分化和存活。配体与EGF受体细胞外结构域的N端残基结合会诱导构象变化、二聚化以及细胞内酪氨酸残基的（自）磷酸化。此外，活化的EGF受体可能对存活途径产生积极影响，从而决定肿瘤生长和进展的途径。值得注意的是，在许多人类恶性肿瘤中，通常会观察到EGF受体活性的过度增强。了解导致EGF受体酪氨酸残基异常磷酸化及其衍生信号级联反应的机制，对于理解癌症发展的分子机制至关重要。在这里，我们总结了最近的标记方法，并讨论了基于质谱的磷酸化分析中用于探测受体酪氨酸激酶（RTK）活性的定量分析的困难。我们还回顾了在样品制备方面的最新进展，以研究膜结合RTK、基于质谱的磷酸肽检测以及对蛋白质标记使用不同定量方法的情况。
