Proteomic Analysis of Notch3 Complexes in Lung Cancer

描述（由申请人提供）：Notch3是一种有趣的质膜结合转录因子，最近与肺癌有关。为了确定Notch3在肺癌中的调控途径，我们建议使用基于质谱的蛋白质组学方法来鉴定和表征肺癌细胞中与Notch3相互作用的蛋白质。我们将使用(a)单特异性抗体或(b)串联亲和纯化（TAP）方案从培养的人肺细胞中恢复选定的Notch3蛋白复合物。纯化的蛋白质复合物将使用高灵敏度质谱法进行鉴定，该方法是在我的赞助商实验室开发的。该方法将微毛细管色谱法与质谱法和基因组数据库分析相结合，直接鉴定纯化蛋白复合物的成分。通过生物信息学分析，鉴定出的被认为与肺癌发展或Notch3功能相关的蛋白将优先进行进一步研究。Notch3与这些候选蛋白的相互作用将通过各种生化方法进行验证。这张已验证的Notch3蛋白相互作用列表将用于模拟控制Notch3对转录事件调节的调节蛋白网络。将选择一组在Notch3转录中有疑似作用的验证蛋白进行Notch3功能研究。我们预计这项研究将有助于确定一些有助于肺癌生物学的新分子事件。此外，在这项工作中开发的方法应该适用于其他靶蛋白和疾病状态，以发现用于诊断或治疗应用的新型蛋白质-蛋白质相互作用。

DESCRIPTION (provided by applicant): Notch3 is an intriguing plasma membrane-bound transcription factor that has recently been implicated in lung cancer. To define the Notch3 regulatory pathway in lung cancer, we propose to use a mass spectrometry-based proteomics approach to identify and characterize proteins that interact with Notch3 in lung tumor cells. We will use either (a) mono-specific antibodies or (b) Tandem Affinity Purification (TAP) protocols to recover selected Notch3 protein complexes from cultured human lung cells. The purified protein complexes will be identified using a high sensitivity mass spectrometry approach developed in my sponsor's laboratory. This approach couples microcapillary chromatography with mass spectrometry and genome database analysis to directly identify components of purified protein complexes. From a bioinformatics analysis, the identified proteins thought to be relevant to lung cancer development or Notch3 function will be given priority for further examination. Notch3 interactions with these candidate proteins will be validated using a variety of biochemical approaches. This list of validated Notch3 protein interactions will be used to model the regulatory protein network controlling Notch3's regulation of transcription events. A subset of validated proteins with suspected roles in Notch3 transcription will be selected for Notch3 functional studies. We anticipate that this study will help define some novel molecular events that contribute to lung cancer biology. Furthermore, the development of methodology in this work should be applicable to other target proteins and disease states for the discovery of novel protein-protein interaction for diagnostic or therapeutic applications.