Hybrid Biological-Abiotic Proximity Labeling Catalysts for Enhancing Spatially-Resolved Proteomics

用于增强空间分辨蛋白质组学的混合生物-非生物邻近标记催化剂

• 批准号: 10717299
• 负责人: Jeffrey Daniel Martell
• 金额: $ 31.42万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10717299
• 关键词: Address; Amino Acids; Benchmarking; Biological; Biological Process; Biotin; Cells; Chemicals; Collaborations; Communities; DNA; Data Set; Detection; Development; Diffuse; Diffusion; Directed Molecular Evolution; Disease; Doctor of Philosophy; Enzymes; Exhibits; Genetic; Goals; Heme; Hybrids; In Vitro; Infection; Kinetics; Label; Laboratories; Length; Location; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Methodology; Methods; Molecular; Nanostructures; Nanotechnology; Nerve Degeneration; Neurons; Pathogenicity; Performance; Peroxidases; Protein-Protein Interaction Map; Proteins; Proteome; Proteomics; Radial; Reaction; Research; Resolution; Sensitivity and Specificity; Shapes; Site; Specificity; Structure; Surface; Synapses; Synaptic Cleft; System; Technology; Tertiary Protein Structure; Tyrosine; cancer cell; catalyst; detection sensitivity; enzyme substrate; improved; interest; nano; nanorod; novel; novel strategies; phenoxy radical; protein function; protein protein interaction; scaffold; synthetic construct; three dimensional structure; tool

A powerful technology for characterizing subcellular proteomes is “proximity labeling” (PL), in which a catalyst is localized to a specific cellular location, followed by promiscuous tagging of endogenous proteins in the vicinity. The tagged proteins are then isolated and identified by mass spectrometry. Although PL catalysts are powerful, new PL catalysts are needed to enhance the sensitivity and specificity of spatially resolved proteomic mapping. Genetically encoded enzymes can be conveniently targeted to cellular locations of interest, but they are limited in their mechanisms of tagging, which hampers control over the labeling radius (limiting specificity) and restricts which amino acids can be tagged (limiting sensitivity). Synthetic PL catalysts have recently introduced a greater diversity of chemical labeling mechanisms, but new approaches are needed for selective activation of these catalysts in highly specific subcellular regions of interest. We propose that hybrid biological-abiotic PL catalysts can achieve improved sensitivity as well as specificity in spatially-resolved proteomic mapping. We are pursuing this hypotheses through the development of three classes of hybrid PL catalysts, which offer complementary advantages. In Aim 1, we have used directed evolution to discover heme peroxidase enzymes capable of generating highly reactive radicals, which exhibit a shorter diffusion radius and label chemically diverse amino acids, in contrast to the APEX approach that almost exclusively labels tyrosines. This ability to react with more amino acids will enhance sensitivity for detecting proximal proteins. In Aim 2, we have developed hybrid DNA- synthetic PL catalysts that become activated only in highly specific subcellular locations. We are applying these switchable catalysts for activation of PL selectively at protein–protein interactions (PPIs) on the surface of cancer cells, and we will extend this approach for activation at intercellular PPIs in neuronal synapses. In Aim 3, we have developed hybrid DNA-synthetic catalysts that tag proteins through contact-dependent mechanisms, instead of generating diffusible reactive species. We will attach these contact-dependent catalysts to DNA nano- rod structures with tunable lengths and rigidities, enabling precise control over the labeling radius in the range of ~1–50 nm. We are applying all three classes of PL catalysts for proteomic mapping in living mammalian cells, in collaboration with Prof. Lloyd Smith, an expert in high-resolution biomolecular mass spectrometry. Additionally, we are collaborating with Prof. Edwin Chapman to employ these PL tools in cultured neurons to benchmark their performance against existing tools. Throughout the next five years, my laboratory will continue to develop new mechanisms for PL using hybrid abiotic-biological catalysts. I envision that these technologies will be employed not only in my laboratory, but also in the broader community, to elucidate novel protein functions in a variety of biological contexts.

鉴定亚细胞蛋白质组的一种强大技术是“邻近标记”(PL)，在这种技术中，催化剂是 定位到特定的细胞位置，然后在附近混杂地标记内源蛋白。 标记的蛋白质随后被分离出来，并通过质谱学进行鉴定。虽然PL催化剂很强大， 需要新的PL催化剂来提高空间分辨蛋白质组图谱的敏感性和特异性。 基因编码的酶可以方便地定位于感兴趣的细胞位置，但它们是有限的 在他们的标记机制中，这阻碍了对标记半径的控制(限制特异性)，并限制了 可以标记哪些氨基酸(限制灵敏度)。合成PL催化剂最近推出了更大的 化学标记机制的多样性，但需要新的方法来选择性地激活这些 具有高度特异性的亚细胞感兴趣区域中的催化剂。我们提出了生物-非生物复合磷光催化剂 可以在空间分辨蛋白质组学图谱中实现更高的敏感性和特异性。我们正在追寻 这一假设通过开发三类杂化PL催化剂，提供了互补性 优势。在目标1中，我们使用定向进化来发现能够 产生高活性的自由基，表现出较短的扩散半径并标记化学上不同的氨基 酸，与APEX方法形成对比，APEX方法几乎完全标记酪氨酸。这种与更多人反应的能力 氨基酸将提高检测近端蛋白质的灵敏度。在目标2中，我们开发了杂交DNA- 仅在高度特定的亚细胞位置激活的合成PL催化剂。我们正在应用这些 肿瘤表面蛋白质-蛋白质相互作用(PPI)选择性激活PL的可切换催化剂 细胞，我们将把这种方法扩展到神经元突触的细胞间PPI激活。在目标3中，我们 已经开发出混合DNA合成催化剂，通过接触依赖机制标记蛋白质， 而不是产生可扩散的活性物种。我们将把这些接触依赖的催化剂连接到DNA纳米- 具有可调长度和刚性的杆结构，可精确控制范围内的贴标半径 ~1-50 nm。我们正在应用所有三种类型的PL催化剂在活的哺乳动物细胞中进行蛋白质组图谱绘制， 与高分辨率生物分子质谱学专家劳埃德·史密斯教授合作。另外， 我们正在与Edwin Chapman教授合作，在培养的神经元中使用这些PL工具来对其进行基准测试 相对于现有工具的性能。在接下来的五年里，我的实验室将继续开发新的 非生物-生物复合催化剂的光致发光机理。我设想这些技术将被应用于 不仅在我的实验室里，而且在更广泛的社区中，阐明各种新的蛋白质功能 生物学背景。