Proteomic Profiling of the Vocal Fold Extracellular Matrix

描述（由申请人提供）：声带细胞外基质（VF-ECM）由复杂的三维蛋白质支架组成，在生物力学上有利于发声。这种蛋白基质的破坏会对声带振荡造成严重后果，通常会导致发声障碍，目前的医学和外科干预措施难以有效治疗。在治疗基于VF-ECM的疾病方面缺乏进展，部分原因是由于对基质的天然生物结构和功能的了解有限，以及它在特定生理和疾病状态下改变的方式。以前的工作已经解决了单个和小群蛋白质在VF-ECM结构和功能中的作用；然而，仍然需要对这些个体如何相互作用以形成功能性生物和生物力学系统有一个总体和统一的理解。VF-ECM研究的蛋白质组学方法提供了同时分析基质内每种蛋白质的方法，并在此过程中代表了系统的整个功能输出。我们的长期目标是构建一个完整的VF-ECM蛋白质组图，阐明伴随特定VF-ECM疾病状态的蛋白质组改变，并构建用于治疗目的的脱细胞和合成VF-ECM支架工程的蛋白质组蓝图。我们的首要长期假设是，VF-ECM的疾病特异性改变是蛋白质组范围变化的结果，涉及细胞介导的功能相互依赖的蛋白质协调。我们的短期假设，我们将在本提案中概述的研究中进行验证，即天然（细胞填充）VF-ECM和脱细胞化VF-ECM具有不同的特征蛋白质组学特征。这项工作将在大鼠模型上完成，并将利用两种互补的蛋白质组学方法：(a)通过二维十二烷基硫酸钠聚丙烯酰胺凝胶电泳（2D SDS-PAGE）分离蛋白质，(b)利用基质辅助激光解吸/电离-飞行时间质谱（MALDI-TOF MS）通过质谱（MS）指纹识别鉴定蛋白质种类。我们的具体目标是：（目标1）建立大鼠VF-ECM蛋白质组的带注释的2D SDS-PAGE参考图；和（目的2）表征和比较天然（细胞填充）VF-ECM和去细胞化VF-ECM的蛋白质组学特征。这些目标的完成将为正在进行的VF-ECM研究提供一个蛋白质组学参考图草案，并为基于脱细胞VF-ECM支架的再生和操作的VF-ECM组织工程策略提供一个蛋白质组学蓝图。这项工作非常重要，因为它将有助于研究任何形式的VF-ECM操作、生理扰动或疾病过程后蛋白质表达的全系统变化；并将直接指导未来声带组织工程的研究。我们预计，在这个项目中产生的制图数据将对其他科学家非常有价值，并包括一个数据共享计划，使我们的原始数据可供科学界使用。

DESCRIPTION (provided by applicant): The vocal fold extracellular matrix (VF-ECM) consists of a complex three-dimensional protein scaffold that is biomechanically favorable for voice production. Disruption of this protein matrix has severe consequences for vocal fold oscillation and generally results in a dysphonia that is difficult to treat effectively with current medical and surgical interventions. Lack of progress in the treatment of VF-ECM based disorders is in part due to limited understanding of the native biological structure and function of the matrix, and the manner in which it is altered under given physiological and disease states. Previous work has addressed the role of individual and small groups of proteins in VF-ECM structure and function; however there remains a need for an overarching and unifying understanding of how these individual players interact to form a functional biological and biomechanical system. A proteomic approach to VF-ECM research offers the means to simultaneously profile every protein within the matrix, and in doing so, represent the entire functional output of the system. Our long-term goal is to construct a fully annotated proteomic map of the VF-ECM, elucidate the proteomic alterations that accompany specific VF-ECM disease states, and construct a proteomic blueprint for the engineering of acellular and synthetic VF-ECM scaffolds for therapeutic purposes. Our overarching long- term hypothesis is that disease specific alterations in the VF-ECM are the result of proteome-wide changes involving the cell-mediated coordination of functionally interdependent proteins. Our short-term hypothesis, which we will test in the research outlined in this proposal, is that the native (cell populated) VF-ECM and decellularized VF-ECM have distinct and characteristic proteomic profiles. This work will be completed using a rat model and will utilize two complementary proteomic methodologies: (a) Protein separation by two- dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE), and (b) protein species identification by mass spectrometry (MS) fingerprinting, utilizing matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS). Our specific aims are: (Aim 1) To establish an annotated 2D SDS-PAGE reference map of the rat VF-ECM proteome; and (Aim 2) to characterize and compare the proteomic profiles of native (cell populated) VF-ECM and decellularized VF- ECM. Completion of these aims will result in a draft proteomic reference map for ongoing VF-ECM research, and a proteomic blueprint for VF-ECM tissue engineering strategies based on the repopulation and manipulation of decellularized VF-ECM scaffolds. This work is highly significant as it will facilitate the investigation of system-wide changes in protein expression following any form of VF-ECM manipulation, physiological perturbation or disease process; and will directly inform future vocal fold tissue engineering efforts. We anticipate that the mapping data generated in this project will be highly valuable to other scientists and have included a data sharing plan to make our raw data available to the scientific community. Project Narrative: This project will result in the development of a research tool that promises to improve our understanding of the network of proteins inside the vocal folds that facilitate vibration for voice production. Using this tool, we will be able to improve our understanding of how the entire network of vocal fold proteins work in concert and how this network is altered by vocal fold disease. This project will also result in the development of a blueprint for the engineering of a healthy protein matrix for restoring damaged vocal folds.