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Proteomic Profiling of the Vocal Fold Extracellular Matrix

声带细胞外基质的蛋白质组学分析

基本信息

批准号：
7850046
负责人：
Nathan Welham
金额：
$ 3.71万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-17 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7850046
关键词：
Address Affect Biological Biomechanics Cells Characteristics Communities Complex Data Databases Development Disease Dysphonia Engineering Extracellular Matrix Fingerprint Funding Future Gel Generations Goals Human Individual Investigation Lamina Propria Lead MALDI-TOF Mass Spectrometry Maps Mass Spectrum Analysis Measurement Measures Mediating Medical Methodology Modeling Operative Surgical Procedures Output Peptides Physiological Process Production Proteins Proteome Proteomics Rattus Research Role Sampling Scaffolding Protein Scientist Silver Staining Sodium Dodecyl Sulfate Spottings Staining method Stains Structure System Testing Therapeutic Tissue Engineering Two-Dimensional Polyacrylamide Gel Electrophoresis Variant Visual Voice Work base data sharing improved innovation interest novel diagnostics protein expression research study scaffold tool two-dimensional vibration vocal cord

项目摘要

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.

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。这种蛋白质基质的破坏对声带振荡具有严重的后果，并且通常导致发声困难，其难以用当前的医学和外科干预有效地治疗。基于VF-ECM的疾病的治疗缺乏进展部分是由于对基质的天然生物学结构和功能以及在给定生理和疾病状态下改变的方式的理解有限。以前的工作已经解决了单个和小群体蛋白质在VF-ECM结构和功能中的作用;然而，仍然需要对这些个体参与者如何相互作用以形成功能性生物学和生物力学系统进行全面和统一的理解。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全注释的VF-ECM蛋白质组图谱，阐明伴随特定VF-ECM疾病状态的蛋白质组改变，并构建一个蛋白质组蓝图，用于治疗目的的无细胞和合成VF-ECM支架的工程设计。我们的总体长期假设是，VF-ECM中的疾病特异性改变是涉及功能上相互依赖的蛋白质的细胞介导的协调的蛋白质组范围的变化的结果。我们的短期假设，我们将在本提案中概述的研究中进行测试，是天然（细胞群）VF-ECM和脱细胞VF-ECM具有不同的和特征性的蛋白质组学谱。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）进行蛋白质分离，和（B）通过质谱（MS）指纹识别，利用基质辅助激光解吸/电离-飞行时间质谱（MALDI-TOF MS）进行蛋白质种类鉴定。我们的具体目标是：（目的1）建立大鼠VF-ECM蛋白质组的注释的2D SDS-PAGE参考图谱;以及（目的2）表征和比较天然（细胞聚集的）VF-ECM和脱细胞VF-ECM的蛋白质组谱。这些目标的完成将导致正在进行的VF-ECM研究的蛋白质组学参考图谱草案，以及基于脱细胞VF-ECM支架的再增殖和操作的VF-ECM组织工程策略的蛋白质组学蓝图。这项工作是非常重要的，因为它将有助于调查蛋白质表达的全系统变化后，任何形式的VF-ECM操作，生理扰动或疾病过程;并将直接通知未来的声带组织工程的努力。我们预计，该项目产生的绘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，使我们的原始数据提供给科学界。项目叙述：该项目将导致一种研究工具的开发，该工具有望提高我们对声带内蛋白质网络的理解，这些蛋白质网络有助于声音产生的振动。使用这个工具，我们将能够提高我们对整个声带蛋白网络如何协同工作以及这个网络如何被声带疾病改变的理解。该项目还将导致开发一种健康蛋白质基质的工程蓝图，用于恢复受损的声带。