Developing gel-based platform for quantitative phosphoproteomics

开发基于凝胶的定量磷酸蛋白质组学平台

• 批准号: 8976425
• 负责人: Anton Iliuk
• 金额: $ 15万
• 依托单位: TYMORA ANALYTICAL OPERATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8976425
• 关键词: Academia; Address; Binding; Biological; Biological Assay; Biological Markers; Clinical; Crosslinker; Data; Data Set; Detection; Development; Diagnostic; Disease; Event; Fractionation; Gel; Goals; Guns; Human; Image; Imagery; Individual; Industry; Ions; Label; Laboratories; Link; Location; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Modification; Pharmacologic Substance; Phase; Phosphopeptides; Phosphoproteins; Phosphorylation; Phosphotransferases; Proteins; Proteomics; Protocols documentation; Reagent; Research Personnel; Role; Running; Sampling; Sensitivity and Specificity; Signal Pathway; Signal Transduction; Small Business Innovation Research Grant; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Techniques; Technology; Therapeutic Agents; Time; Titania; Titanium; Translating; United States National Institutes of Health; anticancer research; base; cancer type; combat; cost; cost effective; design; disease mechanisms study; expectation; experience; fluorophore; gel electrophoresis; imaging software; innovation; innovative technologies; instrument; interest; kinase inhibitor; new technology; new therapeutic target; novel; novel strategies; phase 1 study; phase 2 study; protein complex; public health relevance; research and development; research study; small molecule; tool

 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).

 描述(申请人提供)：随着最近的技术进步，可能导致人类恶性肿瘤的多种重要信号通路不断被发现和解剖。这些信号通路中的绝大多数涉及可逆的蛋白质磷酸化，而有关磷酸化位置和动力学的信息为信号网络如何运作和相互作用提供了重要的机制。虽然质谱学已经成为磷蛋白质组分析的一种特别有用的工具，但对大多数研究人员来说，广泛的实验是非常劳动密集型和成本高昂的。因此，尽管进行了许多大规模的磷蛋白质组学研究，但对相关磷蛋白进行常规和有效分析的迫切需要尚未得到解决。通过这项NIH SBIR第一阶段研究，我们将开发一种新的基于凝胶的磷酸化蛋白质组分析策略，称为差异凝胶电泳法(DiGEP)，并将其转化为商业产品，用于更简单的磷酸化发现分析。这种新颖的设计将利用钛离子功能化的小分子平台选择性地与磷蛋白结合，以及基于紫外光的交联剂将试剂固定在结合的磷蛋白上。此外，两个不同但结构相似的荧光团将被用来区分在单一凝胶上运行的两个样品的磷蛋白质组图谱。该策略将允许对两个样品之间的磷酸化进行定量测量，并将通过质谱学进一步分析对比过的磷蛋白。建议的方法提供了显著降低成本的承诺，并与已经为差异凝胶电泳(DGE)开发的凝胶系统和成像软件完全兼容。