Nanoliter-scale Affinity Purification Mass Spectrometry for Small Numbers of Hair Cells

少量毛细胞的纳升级亲和纯化质谱分析

• 批准号: 10472639
• 负责人: Peter Gordon Barr-Gillespie
• 金额: $ 23.76万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10472639
• 关键词: Adopted; Affinity; Affinity Chromatography; Animal Model; Antibodies; Antibody Dissociations; Auditory system; Benchmarking; Binding; Binding Proteins; Biocompatible Materials; Biological Assay; Biopsy; Cell Separation; Cell physiology; Cells; Chick; Cochlea; Complex; Consumption; Cytolysis; Digestion; Dissociation; Dwarfism; Ear; Ensure; Environment; Goals; Hair Cells; Head Movements; Human; Immobilization; Immunoprecipitation; Inner Hair Cells; Knowledge; Labyrinth; MYO7A gene; Magnetism; Mammalian Cell; Mass Spectrum Analysis; Measurement; Measures; Methods; Microfluidics; Molecular; Mus; Nature; Organ; Performance; Physiological; Preparation; Procedures; Proteins; Proteomics; Recovery; Research; Sampling; Sensitivity and Specificity; Sensory; Surface; System; Technology; Testing; Time; Utricle structure; antigen antibody binding; base; experimental study; hearing loss treatment; improved; innovation; laser capture microdissection; magnetic beads; mechanotransduction; miniaturize; nanoDroplet; nanolitre; nanolitre scale; novel; protein expression; protein protein interaction; sound; theories; therapy development

PROJECT SUMMARY/ABSTRACT Hair cells are the sensory cells of the inner ear that carry out the essential function of mechanotransduction evoked by sound and head movement. It is fundamentally important to characterize global protein expressions and their interactions in hair cells in order to understand the molecular mechanism. However, one of the greatest challenges in protein characterization is the low number of hair cells presented in each inner-ear organ, which urges us to develop sensitive analytical approaches. To this end, our labs develop a microfluidic sample preparation platform, termed as nanoPOTS (nanodroplet processing in one pot for trace samples), for proteomics analysis of low-input biomaterials by downscaling processing volumes to <200 nL. While nanoPOTS is well demonstrated to identify and quantify protein expression from single hair cells, it informs nothing on how proteins interact with each other to implement their functions. The overall objective of this project to develop a sensitive nanoliter droplet-based affinity purification with mass spectrometry (nanoAP-MS) platform to identify protein interacting partners using fewer than 1000 hair cells isolated from utricles or cochleas of the mouse ear. The central hypothesis is that the overall sensitivity of AP-MS assay can be significantly improved by performing affinity purification in nanoliter droplets. Theory suggests this hypothesis should be correct because: 1) Improved protein concentrations by lysing cells in nanoliter volumes will improve protein-bead binding efficiency; 2) Reducing the amounts of affinity beads will reduce non-specific binding, which can otherwise dwarf specific binding; and 3) improved LC-MS will provide sufficient analytical sensitivity to measure low abundance proteins. The central hypothesis will be tested by pursuing two specific aims: 1) To establish a nanoliter droplet-based AP-MS workflow; and 2) To apply this workflow for identification of MYO7A and GIPC3 binding partners in mouse hair cells. We expected the proposed nanoAP-MS platform will increase sensitivity by a factor of 103 or more and allow us to characterize low-abundance protein interaction partners. This research is highly innovative because the nanoAP-MS platform will be the first of its kind to reliably measure protein-protein interactions using a small number of primary cells isolated from physiological environment, including animal models or human biopsies. Statement of Impact: As AP-MS has emerged as powerful technology to discover protein interaction partners and establish protein-protein-interaction networks, the nanoAP-MS technology will enable to examine important protein-protein interactions in small numbers of cells isolated by micropipette, FACS, or laser-capture microdissection, or to examine exceptionally low- abundance interactions like those present in the hair cell's mechanotransduction complex.

项目总结/摘要 毛细胞是内耳的感觉细胞，执行机械传导的基本功能 由声音和头部运动引起。表征蛋白质的全局表达是非常重要的 以及它们在毛细胞中的相互作用，以了解分子机制。不过其中一 蛋白质表征的最大挑战是每个内耳中存在的毛细胞数量少 这份报告敦促我们制定敏感的分析方法。为此，我们的实验室开发了一种微流体 样品制备平台，称为nanoPOTS（用于痕量样品的一锅纳米液滴处理），用于 通过将处理体积缩小到<200 nL，对低输入生物材料进行蛋白质组学分析。而 nanoPOTS被很好地证明可以识别和量化单个毛细胞的蛋白质表达，它告知 没有任何关于蛋白质如何相互作用以实现其功能的信息。本报告的总体目标 项目开发一种灵敏的基于纳升液滴的亲和纯化质谱（nanoAP-MS） 使用少于1000个从椭圆囊分离的毛细胞鉴定蛋白质相互作用伴侣的平台， 老鼠耳朵的耳蜗。中心假设是AP-MS测定的总体灵敏度可以 通过在纳升液滴中进行亲和纯化而显著改善。理论表明这个假设 应该是正确的，因为：1）通过以纳升体积裂解细胞来改善蛋白质浓度将改善 蛋白质-珠子结合效率; 2）减少亲和珠子的量将减少非特异性结合， 其可以使特异性结合相形见绌;和3）改进的LC-MS将提供足够的分析灵敏度 来测量低丰度蛋白质。中心假设将通过追求两个具体目标来检验：1） 建立基于纳升液滴的AP-MS工作流程;以及2）将该工作流程用于鉴定MYO 7A 和GIPC 3结合伴侣。我们预计拟议的nanoAP-MS平台将增加 灵敏度提高了103倍或更多，并使我们能够表征低丰度蛋白质相互作用伴侣。 这项研究是高度创新的，因为nanoAP-MS平台将是同类产品中第一个可靠的 使用从生理分离的少量原代细胞测量蛋白质-蛋白质相互作用 环境，包括动物模型或人类活检。影响声明：由于AP-MS已经成为 发现蛋白质相互作用伙伴和建立蛋白质-蛋白质相互作用网络的强大技术， nanoAP-MS技术将能够检测少量的重要蛋白质-蛋白质相互作用， 通过微量移液器、FACS或激光捕获显微切割分离的细胞，或检查异常低- 丰富的相互作用，就像那些存在于毛细胞的机械转导复合体。