Enabling Top-Down Proteomics through Material Chemistry and Nanotechnology

摘要 在后基因组学时代，对由遗传变异和 翻译后修饰(PTM)对于在功能水平上理解生物系统和 用于解剖复杂的分子系统，并考虑个体差异，用于精确医学。顶端- 基于降质谱(MS)的蛋白质组学是分析完整蛋白质的最有力的方法 全面鉴定蛋白质形式，以破译PTM编码和序列变异。 尽管最近在MS硬件和软件方面都取得了重大进展，以推进自顶向下 MS更接近主流，自上而下的蛋白质组学仍然面临重大挑战。特别是，蛋白质组是 极其复杂，除了许多蛋白质的低溶解度外，还有很高的动态范围，使得它 对高通量蛋白质组学研究具有极大的挑战性。在上一个资助期取得成功的基础上，在本 多PI续展应用，我们将继续开发以纳米技术为动力的创新战略 和材料/有机化学，以进一步应对自上而下的蛋白质组学挑战。具体的 这项建议的目标是：1)为了解决蛋白质的溶解性挑战，我们将开发一种新的策略 由用于细胞外基质(ECM)蛋白质组学的可光裂解表面活性剂实现；以及设计、合成和 评价一类能够保留蛋白质天然结构的新型可光裂解非离子表面活性剂 基于自然MS的自上而下的蛋白质组学。2)为了应对高动态范围的挑战，我们将开发新的 表面功能化磁性纳米颗粒(NPs)模拟抗体捕获和富集Low 丰富的蛋白质，如心肌肌钙蛋白I(cTnI，心脏病的黄金标准生物标记物) 来自细胞/组织的组织/血液和G蛋白偶联受体(GPCRs，一类主要的药物靶标)，用于 通过自上而下的蛋白质组学对所有蛋白质组进行下游综合分析。我们高度跨学科的 该方法将材料化学/纳米技术与自上而下的基于MS的蛋白质组学相结合，并基于 两个PI之间现有的富有成效的合作已取得重大进展，并从 过去的资助期。我们提议的研究的成功将提供创新工具，以实现自上而下 难溶、低丰度蛋白质的蛋白质组学研究，这将为 了解ECM在癌症和心脏疾病的疾病进展中所起的关键作用，定义 天然膜复合体的结构-功能关系，建立一种全面的cTnI测定方法 对心脏疾病的高准确率诊断，并了解gpr信号的重要作用 在包括癌症、糖尿病和心血管疾病在内的多种人类疾病的发病期间。

Summary In the post-genomics era, a comprehensive analysis of “proteoforms” that arise from genetic variations and post-translational modifications (PTMs) is essential for understanding biological systems at a functional level and for dissecting complex molecular systems with consideration of individual variability for precision medicine. Top- down mass spectrometry (MS)-based proteomics that analyzes intact proteins is the most powerful method to comprehensively characterize proteoforms to decipher the PTM codes together with sequence variations. Although significant strides have been made recently in both MS hardware and software to advance top-down MS closer to the mainstream, top-down proteomics still faces major challenges. In particular, the proteome is extremely complex and has a high dynamic range in addition to the low solubility of many proteins, making it highly challenging for high-throughput proteomic study. Building on the success in the last funding period, in this multiple-PI renewal application, we will continue to develop innovative strategies empowered by nanotechnology and materials/organic chemistry to further address the challenges in top-down proteomics. The specific objectives of this proposal are: 1) To address the protein solubility challenge, we will develop a novel strategy enabled by a photocleavable surfactant for extracellular matrix (ECM) proteomics; and design, synthesize, and evaluate a novel class of photocleavable nonionic surfactants that can retain the native structures of proteins for native MS-based top-down proteomics. 2) To address the high dynamic range challenge, we will develop novel surface functionalized magnetic nanoparticles (NPs) to mimic antibodies for capturing and enriching low abundance proteins, such as cardiac troponin I (cTnI, a gold-standard biomarker for heart diseases) from tissues/blood and G-protein coupled receptors (GPCRs, a major class of drug targets) from cells/tissues, for downstream comprehensive analysis of all proteoforms by top-down proteomics. Our highly interdisciplinary approach integrates materials chemistry/nanotechnology with top-down MS-based proteomics, and is based on an existing productive collaboration between two PIs that has led to significant progress and publications from the past funding period. Success in our proposed research will provide innovative tools to enable top-down proteomics of poorly soluble and low abundance proteins, which will lay important technological foundation for understanding the critical role that ECM plays in disease progression in cancer and cardiac diseases, defining the structure-function relationship of native membrane complexes, developing a comprehensive cTnI assay for the diagnosis of cardiac diseases with high accuracy, and understanding the important roles of GPCR signaling during the onset of numerous human diseases including cancer, diabetes, and cardiovascular diseases.