This proposal requests funds for a Thermo Orbitrap Fusion Lumos and an Ultimate 3000 RSLCnano UHPLC instrument to meet increasing demand for high-end mass spectrometry and proteomics instrumentation at the University of Washington. This instrument will be invaluable for the successful completion of existing NIH funded projects requiring sensitive and in-depth characterization of proteins and post-translational modifications. Notably, several of our projects seek to identify quantitative changes in phosphorylation in specific brain regions, requiring the development and implementation of ultra-sensitive new analytical workflows that would be useful for the broader research community. Critical to the successful completion of our NIH research projects is the availability of an integrated system capable of 1) unattended sample injection, true nanoliter flow rates and pressure tolerance to 10,000 psi, 2) sub 1 ppm mass accuracy on a chromatographic time-scale, 3) a dynamic range of > 5000 in a single mass spectrum for accurate quantification and mass determination, 4) rapid high mass resolution scans ~ 450,000 to resolve isobaric mass labels like those in NeuCode SILAC, while 5) acquiring high quality tandem mass spectra (MS2) scans > 15 Hz in parallel with a separate mass analyzer with sensitivity of a quadrupole linear ion trap, 6) synchronous precursor and fragment ion isolation and fragmentation for improved specificity and quantitative accuracy in various quantification methods. Mass spectrometers with these capabilities at the University of Washington are currently heavily oversubscribed and largely unavailable to our grant?s group of users. We propose that the acquisition of the shared proteomics instrument will enable and drive the successful completion of our funded research. The requested instrumentation will allow our faculty to address current technological limitations in their respective fields, improve the sample analysis bottlenecks at the University of Washington, and greatly expand the scope and capabilities of future NIH funded projects.
We are requesting support to purchase a high-end integrated instrument system for qualitative and quantitative analyses of complex protein mixtures. These analyses will help improve our understanding of how our genes are controlled in normal development and disease, study pathways of drug abuse, identify drug targets to kill pathogenic organisms, understand the regulation of ion channels in brain and cardiac muscle, and develop novel clinical assays, and more. All of these areas will improve our understanding, prevention, and treatment of human disease.
|McPherson, Robert Lyle; Ong, Shao-En; Leung, Anthony K L (2018) Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry. Methods Mol Biol 1813:271-283|