Proteomic Analysis of Notch3 Complexes in Lung Cancer

描述(申请人提供)：NOTCH3是一种有趣的质膜结合转录因子，最近被认为与肺癌有关。为了确定肺癌中Notch3的调控途径，我们建议使用基于质谱学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回收选定的Notch3蛋白复合体。纯化的蛋白质复合体将使用我的赞助商实验室开发的高灵敏度质谱学方法进行鉴定。这种方法将微毛细管色谱与质谱仪和基因组数据库分析结合起来，直接鉴定纯化蛋白复合体的成分。从生物信息学分析中，被认为与肺癌发生或Notch3功能相关的已识别蛋白质将优先用于进一步检查。NOTCH3与这些候选蛋白质的相互作用将使用各种生化方法进行验证。这份经过验证的Notch3蛋白相互作用列表将用于对控制Notch3‘S转录事件调控的调控蛋白网络进行建模。在Notch3转录中具有可疑作用的验证蛋白质的子集将被选择用于Notch3功能研究。我们预计，这项研究将有助于定义一些有助于肺癌生物学的新的分子事件。此外，这项工作中方法学的发展应该适用于其他靶蛋白和疾病状态，以发现诊断或治疗应用中新的蛋白质-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.