This proposal requests funds to acquire an ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS) system intended for the identification and structural characterization of nucleic acids, including those containing modified nucleosides. The NIH-funded researchers participating in this request require LC-MS/MS or LC-IMS-MS/MS capabilities to accomplish goals associated with their funded projects. Specifically, the following types of analyses will be enabled by this acquisition: a) Accurate molecular weight measurements of intact nucleic acids;b) Accurate enzymatic mapping of isolated nucleic acids or mixtures of nucleic acids;c) Sequencing of oligonucleotides and enzymatic digestion mixtures by MS/MS;d) Identification of modified nucleosides and their placement within nucleic acid sequences;e) Sequence placement of pseudouridine, the most abundant modified nucleoside, using MRM scans;f) High sensitivity detection and identification of oligonucleotide synthetic side- and by-products;g) Detection of oligonucleotide:peptide cross-links;and h) de novo sequencing of modified RNAs, such as transfer RNAs (tRNAs). The PI, who has been active in the field of mass spectrometry for nearly 20 years, will be responsible for the managerial oversight of the requested instrumentation. He will coordinate oversight with an advisory board composed of scientists from various participating institutions. Together they will establish scheduling priorities, work with accounting personnel to establish cost-recovery rates, and will serve as a resource for projecting future use of the requested equipment. The equipment will be housed in the University of Cincinnati Mass Spectrometry Facility. This 3,500 sq. ft. facility was renovated in 2001 and is equipped with the ancillary instrumentation required for providing the proposed analytical services. The facility's co-directors are two Ph.D.-level staff scientists. One of these scientists will be designated as the primary operator of the new instrument, and it is anticipated that the facility will add a B.S.-level staff technician to ensure that existing and new analytical services are provided in a timely manner.
The UPLC-MS requested in this application will allow NIH-supported researchers access to modern instrumentation necessary for their research programs. These researchers are addressing a variety of health-related issues including: antibiotic resistance, cancer, drug development and nanobiotechnology.
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