Instrumentation for Quantitative Phosphoproteomics and Acetylomics

描述(由申请人提供)：先进的基因组技术使重大的生物医学发现成为可能，而基因组技术的持续技术进步对于更完整的正常和受干扰的生物系统的物理化学表型(定性和定量表征)至关重要。生物系统是极其复杂的，这些系统的物理化学状态的相对微妙的扰动显然会导致生物体健康的深刻变化。杜克蛋白质组学设施(DPCF)的任务是支持医学院7个基础科学系和11个临床科学系的蛋白质/肽表征需求。DPCF为蛋白质鉴定和蛋白质定量提供了基于质谱学的蛋白质组学能力，具有支持各种基础科学研究所需的广泛能力，以及支持临床研究的重大能力，特别是包括生物标记物发现和生物标记物验证实验。DPCF拥有约500万美元的专门分析和信息学硬件/软件，以及7名工作人员(5名博士学位，6名全职工作人员)。DPCF拥有对细胞蛋白质和多肽的LC/LC/MS/MS分析至关重要的管道技术，包括用于蛋白质组、磷蛋白质组和乙酰组学研究的强大的半自动分析管道。为了获得更完整的物理化学表型的磷蛋白质组和乙酰体，DPCF需要额外的最先进的能力和更大的容量，这两者都将由拟议的高分辨率、准确的串联质谱仪(Q-Exactive，Thermo Science)提供。对DPCF和Q-Exactive中相同样品的分析表明，所要求的仪器使乙酰组的覆盖率增加了~9倍，磷酸蛋白质组的覆盖率增加了~7倍(在PTM修饰的多肽水平上)。将DPCF中现有的多维纳米级毛细管超性能UPLC/UPLC系统(Waters NanoAcquity with多维技术)与所需的串联质谱仪(Q-Exactive)相结合，将为磷蛋白质组和乙酰组学分析提供绝对最先进的仪器。这项提议带来的研究项目将促进我们对神经学、肿瘤学、镰状细胞病、传染病和代谢性疾病等领域的疾病和药物治疗机制的理解。最终，该仪器将通过发现和表征健康/疾病/药物反应的蛋白质“生物标记物”来改进临床试验和患者治疗。所要求的系统将提供独特的性能，与DPCF现有的蛋白质组分析系统和全面、专用的信息学基础设施协同一致，使杜克大学能够最大限度地利用广泛的专业知识、强大的数据分析能力以及杜克大学由NIH资助的大量重要的生物医学研究机会。

DESCRIPTION (provided by applicant): Major biomedical discoveries have been made possible with advanced 'omic technologies, and continued technological advances in 'omic technologies are essential for a more complete physicochemical phenotyping (qualitative and quantitative characterizations) of normal and perturbed biological systems. Biological systems are overwhelming complex, and relatively subtle perturbations in the physicochemical status of these systems clearly lead to profound changes in the health of the organism. The Duke Proteomics Facility (DPCF) is tasked with supporting the protein/peptide characterization needs of the seven Basic Science Departments and the eleven Clinical Sciences Departments of the School of Medicine. The DPCF provides capabilities for mass spectrometry based proteomics for protein identification and protein quantitation, with the wide scope of capabilities required t support a variety of basic science studies and the significant scale of capacity to support clinica studies, specifically including biomarker discovery and biomarker verification experiments. The DPCF has ~ $5,000,000 in dedicated analytical and informatic hardware/software, and a staff of 7 (5 with PhDs, 6 of 7 full time staff). The DPCF has the pipeline technologies critical for LC/LC/MS/MS analysis of cellular proteins and peptides, including a robust and semi-automated analysis pipeline for proteomic, phosphoproteomic and acetylomics studies. In order to more completely physiochemically phenotype phosphoproteomes and acetylomes, the DPCF needs both additional state-of-the-art capabilities and increased capacity, both of which would be provided by the proposed electrospray ionization high resolution, accurate mass tandem mass spectrometer (Q-Exactive, Thermo Scientific). Analyses of identical samples in the DPCF and on a Q-Exactive show the requested instrument provides ~9X increase in coverage of the acetylome and ~ 7X increase in coverage of the phosphoproteome (at the PTM-modified peptide level). Coupling an existing multidimensional nanoscale capillary ultraperformance UPLC/UPLC system (Waters NanoAcquity with Multidimensional Technology) in the DPCF and the requested tandem mass spectrometer (Q-Exactive) will provide the absolute state-of-the-art in instrumentation for phosphoproteomic and acetylomics analyses. The research projects enabled by this proposal will advance our understanding of mechanisms of disease and drug therapy in areas including neurology, oncology, sickle cell disease, infectious diseases, and metabolic diseases. Ultimately, the instrumentation will improve clinical trials and patient therapies through the discovery and characterization of protein 'biomarkers' of health/disease/drug response. The requested system will provide unique performance capabilities that are synergistically aligned with the existing proteomic analysis systems and the comprehensive, dedicated informatics infrastructure in the DPCF, enabling Duke to leverage to the fullest extent the extensive expertise, robust data analysis capabilities, and plethora of important NIH- funded biomedical research opportunities at Duke.