Enhanced proteomics capabilities for quantitation of proteins with a triple quad

University Of Cincinnati, Cincinnati OH

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Abstract

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.