Orbitrap Elite Mass Spectrometer for Chromatin Biology and Epigenetics Research

描述（由申请人提供）：本申请申请资助购买赛默飞世尔科学轨道rap精英质谱仪，以支持宾夕法尼亚大学佩雷尔曼医学院表观遗传学项目的新蛋白质组学研究。表观遗传学项目成立于2009年，旨在促进和发展染色质生物学和表观遗传学的基础研究，并组织和鼓励基础和临床科学家之间的合作。表观遗传学计划的几个目标是建立核心研究技术，目前包括功能基因组学，机器人技术和高通量qPCR，染色质和核小体，以及计算表观遗传学。表观遗传学项目建立的最新核心是蛋白质组学资源核心，其中Orbitrap Elite仪器将被安置在PI实验室旁边，以确保最大限度的使用和日常维护。目前，有超过60名NIH资助的教师参与表观遗传学项目，横跨7所不同的费城地区大学，该仪器最初将支持11个主要用户和7个次要用户的研究，这些用户有超过40个NIH资助的活跃项目。该仪器支持的项目包括广泛深入的生物领域和蛋白质组学实验，旨在表征蛋白质丰度和翻译后修饰（PTM）变化。这些研究包括检查代谢和基因调控之间的联系，衰老中涉及的染色质修饰，多能干细胞信号通路，癌症中赖氨酸甲基转移酶活性的改变，询问细胞和染色质网络之间的串扰以及获得单基因蛋白质谱，仅举几例。这台新仪器为这些项目提供了定量蛋白质组学实验，包括组蛋白和非组蛋白组合PTM代码的Top和Middle Down分析，细胞培养中氨基酸的脉冲稳定同位素标记（pulse SILAC），用于确定PTM特异性的转换/动态，通过SILAC和串联质量标记（TMT）进行全球蛋白质组和磷蛋白质组规模的定量，以及含有特殊蛋白质变体的核小体的H/D交换结构实验。等等。由于缺乏适当的仪器或能力，本提案中描述的不同类型的大规模定量蛋白质组学实验要么从未进行过，要么没有在宾夕法尼亚大学校园常规进行。该仪器将使研究与人类健康直接相关，因为表观遗传机制已被证明在许多过程中发挥复杂的作用，例如神经和免疫紊乱以及癌症等疾病。

DESCRIPTION (provided by applicant): This proposal requests funding to purchase a Thermo Fisher Scientific Orbitrap Elite mass spectrometer to support new proteomics efforts in the Epigenetics Program in the Perelman School of Medicine at the University of Pennsylvania. The Epigenetics Program was recently established in 2009 to promote and develop basic research in chromatin biology and epigenetics, and to organize and encourage collaborations between basic and clinical scientists. Among several objectives of the Epigenetics Program is the establishment of core research technologies which currently include Functional Genomics, Robotics and High-throughput qPCR, Chromatin and Nucleosomes, and Computational Epigenetics. The newest core being established by the Epigenetics Program is the Proteomics Resource core where the Orbitrap Elite instrument will be housed next to the laboratory of the PI to ensure maximum usage and routine maintenance. Currently, there are over 60 NIH-funded faculty members in the Epigenetics Program spanning 7 different Philadelphia area universities, and this instrument will initially support the research of 11 major and 7 minor users with over 40 active NIH funded grants. The projects supported by this instrument include a vast depth and breadth of biological areas and proteomics experimentation geared at charactering protein abundance and post-translational modification (PTM) changes. These studies include examining the link between metabolism and gene regulation, chromatin modifications involved in aging, pluripotent stem cell signaling pathways, altered lysine methyltransferase activity in cancer, interrogating the crosstalk between cellular and chromatin networks and obtaining single gene protein profiles, just to name a few. Quantitative proteomic experiments afforded by this new instrument for these projects include Top and Middle Down analyses of histone and non-histone combinatorial PTM codes, pulse stable isotope labeling of amino acids in cell culture (pulse SILAC) for determining PTM-specific turnover/dynamics, global proteome and phosphoproteome scale quantification by SILAC and tandem mass tagging (TMT), and H/D exchange structural experiments on nucleosomes containing specialized protein variants, among others. The different types of large-scale quantitative proteomics experiments described in this proposal either have never been performed or are not routinely performed on the Penn campus due to lack of appropriate instrumentation or capabilities. The studies that will be enabled by this instrument are directly related to human health as epigenetic mechanisms have been shown to play intricate roles in many processes such as neurological and immunological disorders, and diseases such as cancer.