Enhanced proteomics capabilities for quantitation of proteins with a triple quad

增强的蛋白质组学能力，可使用三重四极杆定量蛋白质

• 批准号: 7793821
• 负责人: Kenneth Donald Greis
• 金额: $ 42.62万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-07 至 2011-09-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7793821
• 关键词: Antibodies; Area; Biological; Biological Markers; Clinical; Collaborations; Communities; Complex Mixtures; Diabetes Mellitus; Disease; Elements; Funding; Gel; Heart Diseases; High Pressure Liquid Chromatography; Image Analysis; Ions; Isotopes; Knock-out; Label; Laboratories; Lead; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Measures; Medical center; Methods; Mission; Modeling; Pediatric Hospitals; Peptides; Pharmaceutical Preparations; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Protein Processing; Protein Analysis; Proteins; Proteomics; Request for Applications; Research; Research Personnel; Research Project Grants; Sampling; Services; Site; Sorting - Cell Movement; Technology; Universities; Water; Work; base; comparative; instrument; instrumentation; mass spectrometer; meetings; outcome forecast; patient population; public health relevance; small molecule; validation studies

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.

描述（由申请人提供）：辛辛那提大学（UC）蛋白质组学实验室（PL）提供了广泛的蛋白质组学和生物学质谱（MS）功能。其中包括对蛋白质和肽的常规分析，传统的蛋白质组学方法（比较二-D凝胶分析，图像分析和通过MS鉴定的蛋白质鉴定），蛋白质修饰的映射位点以及使用同位素标记和无标记的MS使用同位素标记的MS进行研究的定量蛋白质组学。在过去的两年中，UC-PL与UC和辛辛那提儿童医院医疗中心（CCHMC）研究社区的50多名调查人员合作，以满足蛋白质组学服务需求并积极从事协作研究项目。 UC-PL目前配备了一个应用生物系统4800 MALDI-TOF/TOF MS，Thermofisher LTQ离子陷阱，带有Eksigent nanolc，一种带有LC-PACKINGE的Thermofisher LCQ离子诱捕器，具有LC-PACKINGS ULTIMATION NANOLC，以及A WATERS LCT LCT LCT LCT LCT lct lct lct ta and an an an an an an an an an anlainciance ht ht ht ht ht ht ht。但是，当前使用现有仪器限制的一种能力是复杂混合物的定量蛋白质测量。最适合定量的最大选择性和敏感性的仪器是三倍的四极质量光谱仪。长期以来，这项技术一直是从复杂混合物中定量测量小分子药物的选择方法，并且最近通常将其用于从复杂混合物中绝对定量蛋白质和肽的绝对定量。 UC和CCHMC研究界有几个现有研究人员，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资助的研究人员使用Seldi-Tof-MS技术来识别疾病状态或预后的生物标志物。但是，他们现在希望通过从临床样本的直接定量中验证这些生物标志物。不幸的是，没有可用的抗体来量化所有候选生物标志物，从而使验证研究不可行。其次，使用特定的激酶或磷酸酶基因敲除模型以识别下游靶标，目前与其他4个由NIH资助的研究者的合作针对全球磷酸化变化。这种全局的磷酸化谱分析导致了许多候选磷酸化位点，但是需要直接定量磷酸化变化才能验证与敲除特有的变化概念，并分类与磷酸化变化相关的基本生物学机制。不幸的是，由于缺乏能够量化蛋白质和磷酸蛋白具有足够敏感性和精确度以测量相关磷酸化的相关调节的高灵敏度仪器，这些定量研究受到了阻碍。因此，该应用程序要求资金购买高选择性/敏感性基于Nanolc的组合三倍四倍/线性离子陷阱仪器（例如，Applied Biosystems 4000 Q Trap）最初支持这7个主要研究人员，但也为整个UC和CCHMC研究社区提供了整个UC和CCHMC研究社区的持续支持。 公共卫生相关性：了解蛋白质变化的机制和蛋白质修饰的通量是理解和调节疾病（例如癌症，心脏病和糖尿病）的关键因素。该仪器将提供准确测量这种蛋白质变化的手段，从而导致这些疾病的新治疗方法。

项目成果

Cardiac metabolic pathways affected in the mouse model of barth syndrome.

• DOI: 10.1371/journal.pone.0128561
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Huang Y;Powers C;Madala SK;Greis KD;Haffey WD;Towbin JA;Purevjav E;Javadov S;Strauss AW;Khuchua Z
• 通讯作者: Khuchua Z

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

• DOI: 10.1016/j.bbrc.2016.10.128
• 发表时间: 2016-11-25
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Kirley TL;Greis KD;Norman AB
• 通讯作者: Norman AB

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

• DOI: 10.1038/s41380-020-0751-3
• 发表时间: 2021-09
• 期刊: Molecular psychiatry
• 影响因子: 11
• 作者: Bentea E;Villers A;Moore C;Funk AJ;O'Donovan SM;Verbruggen L;Lara O;Janssen P;De Pauw L;Declerck NB;DePasquale EAK;Churchill MJ;Sato H;Hermans E;Arckens L;Meshul CK;Ris L;McCullumsmith RE;Massie A
• 通讯作者: Massie A

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE.

• DOI: 10.1016/j.bbrep.2018.10.004
• 发表时间: 2018-12
• 期刊: Biochemistry and biophysics reports
• 影响因子: 2.7
• 作者: Kirley TL;Greis KD;Norman AB
• 通讯作者: Norman AB

Phosphopeptide separation using radially aligned titania nanotubes on titanium wire.

• DOI: 10.1021/acsami.5b00799
• 发表时间: 2015-05
• 期刊: ACS applied materials & interfaces
• 影响因子: 9.5
• 作者: A. Wijeratne;D. Wijesundera;M. Paulose;Ivy Belinda Ahiabu;W. Chu;O. Varghese;K. Greis