Nanotechnology Enabled Top-Down Phosphoproteomics

描述(申请人提供)：蛋白质磷酸化是最常见和最重要的翻译后修饰之一，在许多生物过程中起着关键作用，如细胞生长、分裂和信号传递。磷酸化介导的信号通路的失调与许多疾病有关，如癌症和心脏病。因此，从复杂的生物样品中有效地捕获、分离和综合分析磷蛋白对于理解基本的细胞生物学和疾病机制以及疾病诊断是至关重要的，但仍然是一个重大挑战。在此，我们的目标是通过开发一种智能的多价纳米颗粒(NPs)来从复杂的生物样本中全球浓缩磷蛋白，然后对完整的磷蛋白进行自顶向下的质谱分析，从而解决对磷蛋白质组进行全面分析的挑战。这种跨学科的方法融合了纳米技术和基于自上而下的质谱学的蛋白质组学中令人兴奋的创新，并利用了它们的互补优势。与传统的磷酸蛋白质组学方法相比，它具有显著的优势，包括：由于NPs的高比表面积而具有高捕获能力，由于NPs的类似抗体的多价特性而具有高结合亲和力，高度特异和有效地捕获磷蛋白，与抗体方法不同的是对所有类型的磷蛋白进行普遍的浓缩，保存用于蛋白质功能研究的磷蛋白活性，以及对磷蛋白质组进行全面的表征，并以100%的序列覆盖率定位所有的磷酸化位点。我们将利用磁性或非磁性材料合成多价纳米粒子，并用能够选择性和可逆地与磷酸基团结合的金属螯合配体对其进行功能化。我们已经演示了这项技术的原理证明，并使用我们合成的模型蛋白质混合物和NPs显示了高度特异和有效的浓缩。我们将继续使用模型蛋白质混合物和来自心脏组织的蛋白质来改进这种浓缩方法的性能。捕获的完整磷蛋白将进一步用多维液相色谱分离，并用我们已经开发或正在开发的高分辨率自上而下的质谱仪进行分析。这种基于蛋白质的创新技术用于有效分离和分析完整的磷蛋白，可能会改变主流磷蛋白质组学的范式。这项研究的成功将为整个生物和基础医学研究领域的研究人员，特别是蛋白质组研究人员、酶学家和细胞生物学家提供一项强大的新技术。 与公共健康相关：这项建议寻求开发新型智能纳米材料，用于从复杂蛋白质组中高度特异和高效地在全球范围内捕获磷蛋白，然后对捕获的完整磷蛋白进行全面的蛋白质组分析。这一建议的成功将对通过描绘磷酸化介导的细胞信号通路来理解许多疾病的分子机制产生重大影响，并可能使以磷蛋白为生物标志物的疾病的临床诊断方法的发展成为可能。

DESCRIPTION (provided by applicant): Protein phosphorylation, one of the most common and important post-translational modifications, plays a pivotal role in the control of many biological processes such as cell growth, division, and signaling. Dysregulation of the phosphorylation-mediated signaling pathways has been linked to many diseases such as cancers and heart diseases. Therefore, the effective capture, separation, and comprehensive analysis of phosphoproteins from complex biological samples are crucial for understanding fundamental cell biology and disease mechanisms as well as disease diagnosis, but remain a major challenge. We herein aim to solve the challenge towards a comprehensive analysis of the phosphoproteome by developing a class of smart multivalent nanoparticles (NPs) for enriching phosphoproteins globally out of complex biological samples followed by "top-down" mass spectrometric (MS) analysis of intact phosphoproteins. This interdisciplinary approach integrates the exciting innovations in both nanotechnology and top-down mass spectrometry-based proteomics and capitalizes on their complementary strengths. It has significant advantages over the conventional methods for phosphoproteomics including: high capturing capacity due to high surface area of NPs, high binding affinity due to antibody-like multivalent characteristics of NPs, highly specific and effective capture of phosphoproteins, universal enrichment of all types of phosphoproteins unlike antibody approach, preservation of phosphoprotein activity for protein functional studies, and comprehensive characterization of phosphoproteome and mapping all phosphorylation sites with 100% sequence coverage. We will synthesize the multivalent NPs using magnetic or non-magnetic materials and functionalize them with metal chelate ligands that can selectively and reversibly bind to phosphate groups. We have demonstrated the proof-of-principle of this technology and showed the enrichment is highly specific and effective using model protein mixtures and the NPs we have synthesized. We will continue to improve the performance of this enrichment method using model protein mixtures and proteins from heart tissues. The captured intact phosphoproteins will be further separated by multidimensional liquid chromatography and analyzed by high resolution top-down mass spectrometry we have developed or are developing. This innovative protein-based technology for effective separation and analysis of intact phosphoproteins will potentially transform the paradigm of the main-stream phosphoproteomics. The success of this research will provide a powerful new technology to researchers in the entire biological and basic medical research communities, particularly proteomic researchers, enzymologists, and cell biologists. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop novel smart nanomaterials for highly specific and efficient capturing of phosphoproteins globally out of complex proteome followed by comprehensive proteomic analysis of the captured whole phosphoproteins. The success of this proposal will have significant impacts on understanding molecular mechanisms of many diseases by delineating phosphorylation-mediated cell signaling pathways, and could enable the development clinical diagnosis assays for diseases where phosphoproteins serves as biomarkers.