LTQ-Orbitrap XL for protein identification and biomarker discovery

描述(由申请人提供)：目前的提案将通过提供资金购买具有电子转移解离(ETD)能力的LTQ-Orbitrap XL来帮助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细胞、组织和大分子组装中的蛋白质及其修饰。这将允许检测生物过程或医疗条件所特有的蛋白质表达谱或蛋白质复合体组成的动态变化。该设施和NIH支持的生物医学研究人员之间有许多合作，在这项提案中概述了各种领域，包括先天性免疫系统，寻找糖尿病、前列腺癌和膀胱癌等疾病的生物标记物，以及基因调节、DNA修复、染色体分离和细胞分化的基本机制。这在一定程度上是通过开发一种新的蛋白质组战略来实现的，该战略首先试图全面绘制蛋白质组空间图，从数据库中的蛋白质组图收集数据，并利用先前的信息进行稳健、快速和可重复的蛋白质组分析。这与传统的蛋白质组学方法有很大的不同，在传统的蛋白质组学方法中，在每一次实验中，样品中的蛋白质都是从头开始鉴定的，而不使用其他蛋白质组学实验的先验知识。很明显，这一新的策略对于实现利用质谱学进行全面、定量的蛋白质组分析是必不可少的。获得所要求的仪器将极大地加快我们完成蛋白质组图谱的能力，因为可以预期，难以被CAD拆分的前体将易于被ETD拆分。此外，将高质量准确度和质量分辨率与ETD裂解技术相结合，可以对更大的多肽进行表征，并提高多肽鉴定的置信度。总之，具有ETD的Orbitrap的这些特征将显著增强我们全面描述蛋白质组、亚蛋白质组和单个蛋白质的能力。ISB目前没有ETD技术，在西雅图地区有限地获得具有高质量精度/分辨率并能够执行电子转移反应的仪器将不能满足我们的要求。与公共健康相关的蛋白质在执行几乎所有细胞功能方面发挥着重要作用。因此，能够全面地表征它们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，为理解健康和疾病中细胞功能的分子基础提供了必不可少的信息。所要求的仪器提供的技术将使我们能够实现我们雄心勃勃的全面蛋白质组表征的目标。

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.