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

项目摘要/摘要 毛细胞是内耳的感觉细胞，执行机械转导的基本功能。 由声音和头部运动引起的。确定全球蛋白质表达的特征是非常重要的。 以及它们在毛细胞中的相互作用，以了解其分子机制。然而，其中一个 蛋白质特性的最大挑战是每个内耳中的毛细胞数量很少。 器官，它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控 样品制备平台，称为纳米POTS(痕量样品一锅处理纳米液滴)，用于 低投入生物材料的蛋白质组学分析--将加工量缩减至200nl。而当 它通知说，NanPOTS被很好地证明可以识别和量化单个毛细胞的蛋白质表达 没有关于蛋白质如何相互作用来实现其功能的内容。这样做的总体目标是 开发一种灵敏的纳升液滴亲和纯化-质谱仪(NanAP-MS)的项目 使用不到1000个从胞果或卵胞中分离的毛细胞来识别蛋白质相互作用伙伴的平台 小鼠耳朵的耳蜗虫。中心假设是AP-MS分析的总体灵敏度可以是 通过在纳升液滴中进行亲和纯化，显著提高了检测效率。理论认为这一假说 应该是正确的，因为：1)通过以纳升体积裂解细胞来提高蛋白质浓度将会改善 蛋白质-微珠结合效率；2)减少亲和微球的量将减少非特异性结合， 这可以使特异性结合相形见绌；以及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。核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；2)应用该工作流程鉴定MYO7A 和小鼠毛细胞中的GIPC3结合伙伴。我们预计拟议的NanAP-MS平台将增加 灵敏度提高了103倍或更多，使我们能够确定低丰度蛋白质相互作用伙伴的特征。 这项研究具有很高的创新性，因为纳米AP-MS平台将是此类平台中第一个可靠地 利用从生理性细胞中分离的少量原代细胞来测量蛋白质之间的相互作用 环境，包括动物模型或人类活组织检查。影响声明：由于AP-MS已成为 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， 纳米AP-MS技术将能够在少量的 通过微吸管、流式细胞仪或激光捕获显微切割分离的细胞，或检查异常低的- 丰富的相互作用，就像毛细胞的机械转导复合体中存在的那样。

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.