Nanotechnology Enabled Top-Down Phosphoproteomics

纳米技术实现自上而下的磷酸化蛋白质组学

• 批准号: 8401127
• 负责人: Song Jin
• 金额: $ 20.91万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401127
• 关键词: Affinity; Amino Acid Sequence; Antibodies; Area; Binding; Biological; Biological Assay; Biological Markers; Biological Preservation; Biological Process; Cells; Cellular biology; Characteristics; Communities; Complex; Complex Mixtures; Development; Disease; Dissociation; Electrons; Fourier Transform; Generations; Heart; Heart Diseases; Ligands; Link; Liquid Chromatography; Liquid substance; Magnetism; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Medical Research; Metals; Methods; Modeling; Molecular; Nanotechnology; Peptide Sequence Determination; Performance; Phosphoproteins; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Precipitation; Proteins; Proteome; Proteomics; Reagent; Research; Research Personnel; Research Project Grants; Resolution; Role; Sampling; Signal Pathway; Signal Transduction; Solutions; Stream; Surface; Technology; Time; Tissues; base; cell growth; clinical Diagnosis; design; disease diagnosis; improved; innovation; innovative technologies; inorganic phosphate; interdisciplinary approach; nanomaterials; nanoparticle; new technology; novel; protein aggregation; protein complex; success; tandem mass spectrometry; ultra high resolution

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.

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，在控制许多生物学过程（例如细胞生长，分裂和信号传导）中起着关键作用。磷酸化介导的信号通路的失调与许多疾病（例如癌症和心脏病）有关。因此，对复杂生物样品的磷蛋白的有效捕获，分离和全面分析对于理解基本细胞生物学和疾病机制以及疾病诊断至关重要，但仍然是一个重大挑战。我们本文旨在通过开发一类智能多价纳米颗粒（NP）（NP）来解决对磷酸蛋白酶进行全面分析的挑战，以富集复杂的生物样品中全球磷酸蛋白，然后进行“自上而下的”质谱（MS）分析完整磷酸蛋白。这种跨学科的方法整合了纳米技术和自上而下的质谱蛋白质组学中令人兴奋的创新，并利用其互补优势。它比传统的磷酸蛋白质组学具有显着优势，包括：由于NP的高表面积，高捕获能力，由于NP的抗体样的多价特征，高度特异性的磷酸蛋白，高度特异性捕获，普遍捕获所有类型的磷酸蛋白不与抗体接近所有类型的磷酸化蛋白，并富含Phosplote optraption proptigation optraption proptigation optraption proptigation optraption proptigation rpplote蛋白，因此高度特异性地捕获了磷酸蛋白的高度结合亲和力，高度且有效地捕获了。磷酸化位点具有100％序列覆盖率。 我们将使用磁性或非磁性材料合成多价NP，并使用可以选择性和可逆地结合磷酸基团的金属螯合配体功能化。我们已经证明了这项技术的原理证明，并使用模型蛋白质混合物和我们合成的NPS表明富集是高度特异性和有效的。我们将使用模型的蛋白质混合物和心脏组织中的蛋白质继续提高这种富集方法的性能。捕获的完整磷蛋白将通过多维液相色谱法进一步分离，并通过我们开发或正在开发的高分辨率自上而下的质谱法分析。这项基于创新的蛋白质技术用于有效分离和分析完整的磷蛋白，将有可能改变主流磷酸蛋白质组学的范式。这项研究的成功将为整个生物学和基础医学研究社区的研究人员，尤其是蛋白质组学研究人员，酶学家和细胞生物学家提供强大的新技术。