Developing gel-based platform for quantitative phosphoproteomics

 描述（由申请人提供）：随着最近的技术进步，可能是人类恶性肿瘤原因的多种重要信号通路已被不断发现和剖析。这些信号通路中的绝大多数涉及可逆的蛋白质磷酸化，并且关于磷酸化的位置和动力学的信息提供了关于信号网络如何起作用和相互作用的重要机制。虽然质谱已经成为磷酸化蛋白质组分析的一个非常有用的工具，但对大多数研究人员来说，大量的实验是非常劳动密集型和成本高昂的。因此，尽管许多大规模的磷酸化蛋白质组学研究，一个常规的和有效的分析相关的磷酸化蛋白的关键需求尚未得到解决。通过这项NIH SBIR I期研究，我们将开发一种基于凝胶的磷酸化蛋白质组分析的新策略，称为磷酸化蛋白质组差异凝胶电泳（DiGEP），并将其转化为商业产品，用于更简单的磷酸化发现测定。这种新的设计将利用钛离子功能化的小分子平台，用于选择性结合磷蛋白和基于UV的交联剂，以将试剂固定在结合的磷蛋白上。此外，将使用两种不同但结构相似的荧光团来区分在单个凝胶上运行的两种样品的磷酸化蛋白质组谱。该策略将允许定量测量两个样品之间的磷酸化，并将针点对比磷蛋白应进一步通过质谱分析。所提出的方法提供了显着降低成本的承诺，并完全兼容凝胶系统和成像软件已经开发的差异凝胶电泳（DIGE）。

 DESCRIPTION (provided by applicant): With recent technical advances, multiple important signaling pathways that may be the causes of human malignancy have continuously been discovered and dissected. The vast majority of these signaling pathways involve reversible protein phosphorylation, and the information on the location and dynamics of phosphorylation provides important mechanisms on how the signaling networks function and interact. While mass spectrometry has become an exceptionally useful tool for phosphoproteome analyses, extensive experiments are very labor-intensive and cost-prohibitive to most researchers. As a result, despite many large-scale phosphoproteomic studies, the critical need for a routine and effective analysis of relevant phosphoproteins has not been addressed. Through this NIH SBIR Phase I study, we will develop a novel strategy for gel-based phosphoproteome analysis, called Difference Gel Electrophoresis of Phosphoproteome (DiGEP), and translate it into commercial products for simpler phosphorylation discovery assays. The novel design will take advantage of the small molecule platform functionalized with titanium ions for selective binding to phosphoproteins and a UV-based crosslinker to immobilize the reagent onto the bound phosphoprotein. In addition, two different but structurally-similar fluorophores will be used to differentiate the phosphoproteome profiles of two samples ran on a single gel. The strategy will allow quantitative measurements of phosphorylation between the two samples and will pin-point which contrasted phosphoproteins should be further analyzed by mass spectrometry. The proposed approach offers the promise of significant cost reduction and is fully compatible with gel systems and imaging software already developed for Difference Gel Electrophoresis (DIGE).