Nanotechnology Enabled Top-Down Phosphoproteomics

描述（由申请人提供）：蛋白质磷酸化是最常见和最重要的翻译后修饰之一，在许多生物过程如细胞生长、分裂和信号传导的控制中起着关键作用。磷酸化介导的信号通路失调与许多疾病如癌症和心脏病有关。因此，从复杂的生物样品中有效地捕获、分离和综合分析磷酸化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至关重要，但仍然是一个主要的挑战。我们在此旨在通过开发一类智能多价纳米颗粒（NPs）来解决对磷蛋白质组进行全面分析的挑战，该纳米颗粒用于从复杂的生物样品中富集整体磷蛋白，然后对完整的磷蛋白进行“自上而下”的质谱（MS）分析。这种跨学科的方法整合了纳米技术和自上而下的基于质谱的蛋白质组学的令人兴奋的创新，并利用了它们的互补优势。与传统的磷蛋白质组学方法相比，它具有显著的优势，包括：由于NPs的高表面积而具有高捕获能力，由于NPs的抗体样多价特性而具有高结合亲和力，磷酸化蛋白的高度特异性和有效捕获，与抗体方法不同的是，所有类型的磷酸化蛋白普遍富集，保留磷酸化蛋白活性用于蛋白质功能研究，以及全面表征磷酸化蛋白组并以100%的序列覆盖率绘制所有磷酸化位点。我们将使用磁性或非磁性材料合成多价NPs，并使用金属螯合配体将其功能化，这些配体可以选择性地和可逆地与磷酸基团结合。我们已经证明了该技术的原理证明，并表明富集是高度特异性和有效的，使用模型蛋白混合物和我们合成的NPs。我们将继续使用模型蛋白混合物和心脏组织蛋白来提高这种富集方法的性能。捕获的完整磷酸化蛋白将通过多维液相色谱进一步分离，并通过我们已经开发或正在开发的高分辨率自上而下的质谱分析。这种基于蛋白质的创新技术可以有效地分离和分析完整的磷蛋白，这将有可能改变主流磷蛋白组学的模式。这项研究的成功将为整个生物学和基础医学研究界的研究人员，特别是蛋白质组学研究人员、酶学家和细胞生物学家提供一项强大的新技术。

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.