Proteomics and Metabolomics Shared Resource

项目摘要-蛋白质组学和代谢组学共享资源 蛋白质组学和代谢组学共享资源(PMSR)的主要目标是提供全面的 为癌症中心成员以及其他区域NCI指定的成员提供蛋白质组学和代谢组学服务 癌症中心(福克斯·蔡斯癌症中心、托马斯·杰斐逊大学癌症中心)，其次是 其他学术机构的研究人员。2017年12月前，主要质谱学(MS) PMSR中的仪器包括Thermo Q Exactive HF、Thermo Q Exactive Plus和Sciex QTRAP 5500混合式三重四极质谱计。这些仪器具有纳米超高压PLC(超高压 在前端带有冷却的自动取样器以进行蛋白质组学分析。传统的流程 代谢物分析采用超高效液相色谱仪。这些LC-MS系统代表了一些性能最高的系统， 目前最灵敏的仪器。PMSR将继续提供常规的蛋白质组服务，如 用LC-MS/MS从凝胶条带或下拉实验中鉴定蛋白质，并通过 电喷雾电离。在上一个预算周期，PMSR越来越多地参与到更具挑战性的工作中 蛋白质组学问题，特别是使用标记对蛋白质组或亚蛋白质组进行全球定量比较- 自由定量(积分MS离子流)，使用稳定同位素，如SILAC或等压标记进行定量 (TMT或iTRAQ)，以及对磷酸蛋白质组、泛素体和乙酰体的深入全球分析。PMSR 将继续提高其深入全面分析全球蛋白质组和 翻译后修饰。PMSR还响应了癌症中心日益增长的需求 癌症代谢领域的成员，通过建立大约180个目标相对定量 极性代谢物。为了进一步提高其代谢组学分析能力，PMSR申请并通过了 最近获得了S10共享仪器拨款(S10 OD023586)。这笔赠款与癌症一起 中心资金用于购买一台高分辨率Thermo Q Exactive HF-X质谱仪和一台 征服超高效液相色谱系统。该仪器于2017年12月安装，将专门用于 (全球)代谢组学、靶向代谢组学、非靶向类脂组学、靶向类脂组学和代谢流量 使用稳定的同位素示踪剂进行分析。PMSR最近还购买了一台海马XFe96分析仪，由 另一笔S10赠款(S10OD023658)。该仪器可定量测定线粒体呼吸、糖酵解 活性、内源和外源脂肪酸氧化、底物氧化和代谢表型， 为新的代谢组学LC-MS/MS系统提供补充分析能力。是否可以使用 这些新仪器将确保PMSR在提供蛋白质组学方面保持最先进的水平， 代谢组学，以及为癌症中心成员提供的脂质组学服务。PMSR的主要重点将是 提供专家咨询和最先进的蛋白质组学和代谢组学技术 以最高的性能和实惠的成本解决复杂的癌症生物学问题。

PROJECT SUMMARY – PROTEOMICS AND METABOLOMICS SHARED RESOURCE The primary goal of the Proteomics and Metabolomics Shared Resource (PMSR) is to provide comprehensive proteomics and metabolomics services to Cancer Center members as well as other regional NCI-designated Cancer Centers (Fox Chase Cancer Center, Thomas Jefferson University Cancer Center), and secondarily to investigators in other academic institutions. Prior to December 2017, major mass spectrometry (MS) instruments in the PMSR included a Thermo Q Exactive HF, a Thermo Q Exactive Plus, and a Sciex QTRAP 5500 hybrid triple quadrupole mass spectrometer. These instruments have nano-UHPLCs (ultra-high pressure liquid chromatography) with cooled-autosamplers on the front end for proteomic analyses. A conventional flow UHPLC is used for metabolite analyses. These LC-MS systems represent some of the highest performing, most sensitive instruments currently available. The PMSR will continue to offer routine proteomic services such as LC-MS/MS protein identifications from gel bands or pull-down assays, and intact protein analysis by electrospray ionization. Over the last budget cycle, the PMSR has increasingly engaged in more challenging proteomics problems, particularly global quantitative comparisons of proteomes or sub-proteomes using label- free quantitation (integrated MS ion currents), quantitation using stable isotopes such as SILAC or isobaric tags (TMT or iTRAQ), and in-depth global analysis of phosphoproteome, ubiquitomes, and acetylomes. The PMSR will continue to improve its analytical capabilities for in-depth comprehensive analysis of global proteome and posttranslational modifications. The PMSR has also responded to the growing need of Cancer Center members in the field of cancer metabolism by establishing targeted relative quantitation of approximately 180 polar metabolites. To further increase its capacity in metabolomics analysis, the PMSR applied for and was recently awarded an S10 shared instrumentation grant (S10 OD023586). This grant together with Cancer Center funds were used to purchase a high-resolution Thermo Q Exactive HF-X mass spectrometer and a Vanquish UHPLC system. This instrument was installed in December 2017 and will be dedicated to untargeted (global) metabolomics, targeted metabolomics, untargeted lipidomics, targeted lipidomics and metabolic flux analysis using stable isotope tracers. The PMSR also recently purchased a Seahorse XFe96 Analyzer funded by another S10 grant (S10 OD023658). This instrument can quantify mitochondrial respiration, glycolytic activity, endogenous and exogenous fatty acid oxidation, substrate oxidation, and metabolic phenotype, providing complementary analytical capacity to the new metabolomics LC-MS/MS system. The availability of these new instruments will ensure that the PMSR remains state-of-the-art in providing proteomics, metabolomics, and lipidomics services to Cancer Center members. The major emphasis of the PMSR will be to provide expert consultation and state-of-the-art proteomics and metabolomics technologies operating at maximum performance and affordable costs to address complex cancer biology questions.