Enhanced proteomics capabilities for quantitation of proteins with a triple quad

描述（由申请人提供）：辛辛那提大学（UC）蛋白质组学实验室（PL）提供广泛的蛋白质组学和生物质谱（MS）能力。这些方法包括蛋白质和肽的常规分析，传统的蛋白质组学方法（比较2-D凝胶分析，图像分析和蛋白质鉴定的MS），蛋白质修饰的定位，以及研究导向的定量蛋白质组学使用同位素标记和无标记MS。在过去的2年中，UC-PL已经与来自加州大学和辛辛那提儿童医院医学中心（CCHMC）的50多名研究人员合作研究界，以满足蛋白质组学服务的需求，并积极寻求合作研究项目。UC-PL目前配备了Applied Biosystems 4800 MALDI-TOF/TOF MS、配备Eksigent nanoLC的ThermoFisher LTQ离子阱、配备LC-Packings Ultimate nanoLC的ThermoFisher LCQ离子阱以及配备Alliance HT HPLC的沃茨LCT电喷雾-TOF MS。然而，一个能力，目前是有限的与现有的仪器是定量蛋白质测量复杂的混合物。最适合于最大选择性和灵敏度定量的仪器是三重四极质谱仪。该技术长期以来一直是从复杂混合物中定量测定小分子原料药的首选方法，最近已常规应用于从复杂混合物中绝对定量测定蛋白质和肽。UC和CCHMC研究社区目前有几名研究人员，他们的研究可以从这项技术中受益匪浅。首先，在临床生物标志物领域，3名NIH资助的研究人员已经使用SELDI-TOF-MS技术来识别疾病状态或预后的生物标志物;然而，他们现在希望通过从更大的患者人群的临床样本中直接定量来验证这些生物标志物。不幸的是，没有合适的抗体可用于量化所有候选生物标志物，因此使验证研究不太可行。其次，目前与其他4个NIH资助的研究人员的合作是针对全球磷酸化的变化，使用特定的激酶或磷酸酶敲除模型，以确定下游目标。这种全局磷酸化分析导致了许多候选磷酸化位点，但需要直接定量磷酸化变化来验证敲除特异性的变化并整理出与磷酸化变化相关的潜在生物学机制。不幸的是，这些定量研究受到缺乏高灵敏度仪器的阻碍，该仪器能够以足够的灵敏度和精度定量蛋白质和磷蛋白以测量磷酸化的相关调节。因此，本申请要求提供资金，以购买高选择性/灵敏度的基于纳米LC的组合三重四极杆/线性离子阱仪器（例如Applied Biosystems 4000 Q TRAP），以最初支持这7个主要研究者，但也为整个UC和CCHMC研究社区提供持续支持，作为整体蛋白质组学使命的一部分。 公共卫生相关性：了解蛋白质变化的机制和蛋白质修饰的流量是理解和调节疾病的关键要素，如癌症，心脏病和糖尿病。这种仪器将提供准确测量这种蛋白质变化的方法，这可能导致这些疾病的新治疗方法。

DESCRIPTION (provided by applicant): The University of Cincinnati (UC) Proteomics laboratory (PL) offers a broad array of proteomics and biological mass spectrometry (MS) capabilities. These include routine analysis of proteins and peptide, traditional proteomics approaches (comparative 2-D gels profiling, image analysis and protein identification by MS), mapping sites of protein modification, and research oriented quantitative proteomics using both isotope-tagged and label-free MS. Over the past 2 years, the UC-PL has worked with over 50 investigators across UC and Cincinnati Children's Hospital Medical Center (CCHMC) research community to meet the proteomics service needs and to actively pursue collaborative research projects. The UC-PL is currently equipped with an Applied Biosystems 4800 MALDI-TOF/TOF MS, a ThermoFisher LTQ ion trap with an Eksigent nanoLC, a ThermoFisher LCQ ion trap with an LC-Packings Ultimate nanoLC, and a Waters LCT electrospray-TOF MS with an Alliance HT HPLC. However, one capability that is currently limited with the existing instrumentation is quantitative protein measurement from complex mixtures. The instrument best suited for maximum selectivity and sensitivity for quantitation is a triple quadrupole mass spectrometer. This technology has long been the method of choice for quantitative measurement of small molecule drug substances from complex mixtures and has more recently been routinely applied to absolute quantitation of proteins and peptides from complex mixtures. The UC and CCHMC research community has several current investigators whose research could benefit tremendously from this technology. First, in the area of clinical biomarkers, 3 NIH-funded investigators have used SELDI-TOF-MS technologies to identify biomarkers of disease state or prognosis; however, they now wish to validate these biomarkers by direct quantification from the clinical samples across a much larger patient population. Unfortunately suitable antibodies are not available to quantify all candidate biomarkers thus making the validation study less feasible. Secondly, current collaborations with 4 other NIH-funded investigators are targeted at global phosphorylation changes using specific kinase or phosphatase knockout models to identify downstream targets. This global phosphorylation profiling has led to a number of candidate phosphorylation sites, but direct quantitation of the phosphorylation changes are needed to both validate the changes as specific to the knockout and to sort out the underlying biological mechanisms associated with the phosphorylation changes. Unfortunately these quantitative studies have been hampered by the lack of a high sensitivity instrument capable of quantifying proteins and phosphoproteins with enough sensitivity and precision to measure relevant modulation of phosphorylation. As such, this application requests funding to purchase a high selectivity/sensitivity nanoLC-based combination triple quadrupole/linear ion trap instrument (e.g. an Applied Biosystems 4000 Q TRAP) to initially support these 7 major investigator, but to also provide ongoing support for the entire UC and CCHMC research community as part of the overall proteomics mission. PUBLIC HEALTH RELEVANCE: Understanding mechanism of protein changes and the flux of protein modification are key elements to understanding and modulating diseases, such as cancer, heart disease and diabetes. This instrumentation will provide the means to accurately measure such protein changes that could lead to new treatments of these diseases.