Proteomic Profiling of the Vocal Fold Extracellular Matrix

描述(申请人提供)：声带细胞外基质(VF-ECM)由一种复杂的三维蛋白质支架组成，在生物力学上有利于发声。这种蛋白质基质的破坏会对声带振荡造成严重的后果，通常会导致发音困难，而目前的内科和外科治疗很难有效地治疗这种发音困难。在治疗基于VF-ECM的疾病方面缺乏进展的部分原因是对基质的天然生物结构和功能以及在给定的生理和疾病状态下它的改变方式的了解有限。以前的工作已经解决了单个和小组蛋白质在VF-ECM结构和功能中的作用；然而，仍然需要对这些单个参与者如何相互作用形成一个功能性的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中的每一种蛋白质的方法，并在这样做的过程中，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全注释的VF-ECM蛋白质组图，阐明伴随特定VF-ECM疾病状态的蛋白质组变化，并构建用于治疗目的的无细胞和合成VF-ECM支架的蛋白质组蓝图。我们最重要的长期假设是，VF-ECM中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的协调。我们的短期假设，我们将在这项提案中概述的研究中进行验证，即天然的(细胞填充的)VF-ECM和去细胞的VF-ECM具有不同的和特征的蛋白质组学特征。这项工作将使用大鼠模型完成，并将利用两种互补的蛋白质组方法：(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，以及(B)利用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)进行蛋白质种类鉴定。我们的具体目标是：(1)建立大鼠VF-ECM蛋白质组的注释2D-SDS-PAGE参考图谱；(2)鉴定和比较天然(细胞填充的)VF-ECM和去细胞的VF-ECM的蛋白质组学特征。这些目标的完成将导致正在进行的VF-ECM研究的蛋白质组参考图谱草案，以及基于脱细胞VF-ECM支架的再繁殖和操作的VF-ECM组织工程策略的蛋白质组蓝图。这项工作具有非常重要的意义，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系统范围内蛋白质表达的变化；并将直接为未来的声带组织工程努力提供信息。我们预计，该项目产生的测绘数据将对其他科学家具有很高的价值，并已包括一项数据共享计划，以便将我们的原始数据提供给科学界。 项目描述：该项目将导致开发一种研究工具，有望提高我们对声带内蛋白质网络的理解，该网络有助于声音产生的振动。使用这个工具，我们将能够更好地理解整个声带蛋白质网络是如何协同工作的，以及这个网络是如何被声带疾病改变的。该项目还将为设计修复受损声带的健康蛋白质基质制定蓝图。

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.