LTQ-Orbitrap XL for protein identification and biomarker discovery

描述（由申请人提供）：目前的提案将有助于通过提供资金购买具有电子转移解离（ETD）能力的LTQ-Orbitrap XL来扩大系统生物学研究所（ISB）的蛋白质组学设施。目前，蛋白质组学设施的重点是应用新的方法来系统地表征和量化蛋白质及其在细胞，组织和大分子组装中的修饰。这将允许检测蛋白质表达谱或蛋白质复合物组成的动态变化，其是生物过程或医学状况的特征。该设施与NIH支持的生物医学研究人员之间存在许多合作，这些合作在本提案中概述，涵盖了先天免疫系统，寻找糖尿病，前列腺癌和膀胱癌等疾病的生物标志物，以及基因调控，DNA修复，染色体分离和细胞分化的基本机制等领域。这是通过开发一种新的蛋白质组学策略来实现的，该策略首先尝试全面绘制蛋白质组学空间，从数据库中的蛋白质组图谱收集数据，并使用先验信息进行稳健、快速和可重复的蛋白质组学分析。这代表了与传统蛋白质组学方法的显著偏离，在传统蛋白质组学方法中，在每个实验中，样品中的蛋白质被从头鉴定，而不使用来自其他蛋白质组学实验的先验知识。很明显，新的策略是必不可少的，以达到全面的，定量的蛋白质组分析使用质谱的目标。获得所要求的仪器将大大加快我们完成蛋白质组图谱的能力，因为可以预期，难以通过CAD片段化的前体将易于通过ETD片段化。此外，高质量准确度和质量分辨率与ETD片段化技术的结合使得能够表征更大的肽并提高肽鉴定的置信度。总之，Orbitrap with ETD的这些特性将显著增强我们全面表征蛋白质组、亚蛋白质组和单个蛋白质的能力。ISB目前没有ETD技术，并且在西雅图地区，对具有高质量精度/分辨率并能够进行电子转移反应的仪器的有限访问将不符合我们的要求。蛋白质在执行几乎所有的细胞功能中起着重要作用。因此，全面表征其在正常和扰动系统中的动态表达水平、修饰状态和分子相互作用的能力提供了对于理解健康和疾病中细胞功能的分子基础至关重要的信息。所要求的仪器提供的技术，将使我们能够实现我们的雄心勃勃的目标，全面的蛋白质组表征。

DESCRIPTION (provided by applicant): The current proposal will help to expand the Institute for Systems Biology's (ISB's) Proteomics facility by providing capital to purchase an LTQ-Orbitrap XL with electron transfer dissociation (ETD) capability. Currently the Proteomics facility's focus is on the application of novel approaches to systematically characterize and quantify proteins and their modifications in cells, tissues, and in macromolecular assemblies. This will allow for the detection of dynamic changes in protein expression profiles or protein complex composition that are characteristic for biological processes or medical conditions. Numerous collaborations exist between the facility and NIH supported biomedical researchers that are outlined in this proposal covering areas as diverse as the innate immune system, the search for biomarkers of disease such as diabetes, prostate and bladder cancer, and basic mechanisms of gene regulation, DNA repair, chromosome segregation, and cellular differentiation. This is being accomplished, in part, through the development of a novel proteomic strategy that first attempts to comprehensively map the proteomic space, to collect the data from proteome mapping in a database and to use the prior information for robust, fast and reproducible proteome analysis. This represents a significant departure from the traditional proteomics approaches in which in every experiment the proteins in a sample are identified de novo without using prior knowledge from other proteomics experiments. It has become apparent that the novel strategy is essential to reach the goal of comprehensive, quantitative proteome analysis using mass spectrometry. Access to the requested instrument would greatly accelerate our ability to complete the proteome maps because it can be expected that precursors that are difficult to fragment by CAD would be amenable to fragmentation by ETD. Furthermore, the combination of high mass accuracy and mass resolution with ETD fragmentation technology enables the characterization of larger peptides and improves the confidence of peptide identification. Altogether, these characteristics of the Orbitrap with ETD will significantly enhance our ability to comprehensively characterize proteomes, subproteomes and individual proteins. ETD technology is not currently available at ISB and the limited access to instruments that possess high mass accuracy/resolution with the ability to perform electron transfer reactions in the Seattle area will not meet our requirements. PUBLIC HEALTH RELEVANCE Proteins play a major role in executing virtually all cellular functions. As such, the ability to comprehensively characterize their dynamic expression levels, modification states, and molecular interactions in normal and perturbed systems provides information that is essential to understand the molecular underpinnings of cellular function in health and disease. The requested instrumentation provides the technology that will allow us to attain our ambitious goal of comprehensive proteome characterization.