Nanoparticle based phosphoprotein enrichment for the analysis of phosphoproteins

描述(申请人提供)：蛋白质磷酸化是一种关键的翻译后修饰，细胞用来调节许多生物过程，包括信号传递、生长和分裂。与其在细胞功能中的作用一致，异常的蛋白磷酸化与包括癌症和心脏病在内的许多疾病有关。然而，有效捕获和分析蛋白质组范围的磷酸化模式仍然是一个尚未解决的问题。在这项NRSA博士后奖学金(F32)提案中，我们的目标是通过开发一类多价纳米颗粒材料来解决对蛋白质磷酸化进行全蛋白质组分析的挑战，该材料能够从复杂的生物样本中选择性地浓缩磷蛋白，然后对浓缩的完整蛋白质进行自上而下的蛋白质组学分析。我们的方法结合了纳米颗粒化学和基于质谱学的蛋白质组学的最新进展，并通过提供一个廉价的平台来挑战或改进现有的传统方法，该平台表现出：1)高捕获能力、效率和选择性，2)所有磷蛋白的通用结合，3)可伸缩性和可重用性，以及4)在浓缩后保持天然活性的能力。为了进行浓缩，我们将合成直径为&lt；10 nm的金属铁氧体磁性纳米颗粒，该纳米颗粒带有磷酸特定的结合基团，可以选择性和可逆地结合磷蛋白。捕获和浓缩后，完整的蛋白质将被释放和纯化，并通过高分辨率质谱仪进行分析。通过系统地研究蛋白质颗粒的表面化学和对照(磷酸)蛋白质混合物上的结合/洗脱缓冲条件，将优化蛋白质浓缩的化学。为了研究该平台在生物样本上的能力，我们将演示它在人类心脏蛋白质组分析中的使用，其中细丝蛋白磷酸化对肌肉功能至关重要。这一建议的成功不仅将提供多样化和多学科的研究经验，还将为研究和医学界提供一种强大且易于使用的技术，用于生物样品中蛋白质磷酸化的分析。 与公共卫生相关：这项提议寻求开发一种新的多价纳米材料，用于选择性和有效地从复杂的生物混合物中捕获磷蛋白，然后对浓缩的完整磷蛋白进行全面的蛋白质组学分析。这项工作的成功将在医疗和临床诊断领域产生重大影响，在医疗和临床诊断领域，蛋白质磷酸化可以作为特定情况或疾病的生物标志物，并通过允许表征基于磷酸化的细胞信号通路，为理解许多疾病的分子化学提供有价值的工具。

DESCRIPTION (provided by applicant): Protein phosphorylation is a critical post-translational modification that cells use to regulate many biological processes, including signaling, growth and division. Consistent with its role in cellular function, aberrant protein phosphorylation has been implicated in many diseases including cancer and heart disease. However, efficient capture and analysis of proteome-wide phosphorylation patterns remains an unsolved problem. In this NRSA postdoctoral fellowship (F32) proposal, we aim to solve the challenges of performing a proteome-wide analysis of protein phosphorylation by developing a class of multivalent nanoparticle materials capable of selectively enriching phosphoproteins out of a complex biological sample followed by top-down proteomic analysis of the enriched intact proteins. Our approach combines recent advances in both nanoparticle chemistry and mass-spectrometry-based proteomics and challenges or improves on existing conventional methods by offering an inexpensive platform which exhibits: 1) high capturing capacity, efficiency, and selectivity, 2) universal binding of all phosphoproteins, 3) scalability and reusability, and 4) the ability to preserve native activity following enrichment. For the enrichment we will synthesize metal ferrite magnetic nanoparticles with diameters <10 nm functionalized with phosphate-specific binding groups which can selectively and reversibly bind phosphoproteins. Following capture and enrichment, intact proteins will be released and purified by liquid chromatography and analyzed by high-resolution mass spectrometry. The chemistry of protein enrichment will be optimized by systematically studying the surface chemistry of the particles and binding/elution buffer conditions on a control mixture of (phospho)proteins. To investigate the capabilities of the platform on a biological sample, we will demonstrate its use on the analysis of the human heart proteome where filament protein phosphorylation is critical to muscle function. The success of this proposal will not only provide a diverse and multidisciplinary research experience, it will also provide a powerful and easy to use technology for both the research and medical communities for the analysis of protein phosphorylation in biological samples. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop a new multivalent nanomaterial for the selective and efficient capture of phosphoproteins from complex biological mixtures followed by comprehensive proteomic analysis of enriched intact phosphoproteins. The success of this work will have significant impacts in the medical and clinical diagnostic communities where protein phosphorylation may serve as a biomarker for a specific condition or disease, and will provide a valuable tool for understanding the molecular chemistry of many diseases by allowing the characterization of phosphorylation-based cell signaling pathways.