Multiplex Protein Quantification Core

  • Richardson, Arlan G (PI)

Project: Research project

Project Details

Description

? DESCRIPTION (provided by applicant): Because proteins are critical for all biochemical reactions that occur in living organisms, changes in the levels of proteins in metabolic pathways are important in the etiology of the major diseases facing Americans and veterans. Therefore, having a detailed knowledge of how proteins in biochemical pathway change are key to understanding the molecular basis of diseases and developing therapies to treat the diseases. The Q Exactive LC-tandem mass spectrometer will allow the Oklahoma City VA-Medical Center to develop a Multiplexing Protein Quantification Core, which will use the latest multiplex-technology to target specific proteins in various pathways for quantification. Targeting is achieved by selectively measuring the abundance of several peptides, which are unique to that protein when it is digested by trypsin. These unique peptides can then be detected in complex protein mixtures based on their sequence-specific-fragmentation reactions driven by collision-induced dissociation. Selected Reaction Monitoring is the most well-known method to measure the levels of specific peptides; however, recently developed variations, such as Parallel Reaction Monitoring and Selected Ion Monitoring, are now possible with new high resolution high mass accuracy instruments like the Q Exactive. The peptides are detected as chromatographic peaks at characteristic retention times and the abundance calculated from the area of those peaks. Importantly, the acquisition speed of the mass spectrometer allows the measurement of a large number of peptides in a single mass spectrometry run. The number of proteins measured depends on user choices, but the Multiplexing Protein Quantification Core will routinely quantify panels of 30 to 35 proteins by
detecting 90 to 100 peptides in a single analysis. Thus, using the Q Exactive mass spectrometer, we will be able to measure simultaneously ~30 proteins in a pathway, and because the assay requires small amounts of sample, one can easily measure hundreds of proteins from a single sample. A key to the targeted assay is the need for an advanced LC-tandem mass spectrometry system with nanoflow HPLC and nanospray ionization. The mass spectrometer we are requesting is a Q Exactive Plus from Thermo Electron with a nanospray ion source and Dionex Ultimate 3000 RSLCnano LC system. This mass spectrometer system has the resolution and mass accuracy that are needed to precisely select the parent and fragment ions in the selected reactions as well as the rapid data acquisition rate needed to move between 50-100 reactions per second. Based on our experience with mass spectrometers, this system will give us the resolution and accuracy that we will need in the Multiplexing Protein Quantification Core as well as giving us a mass spectrometer at a reasonable price. The major advantages of targeted protein quantification, which are particularly important for the Multiplexing Protein Quantification Core are: (a) it is readily available to a large number of VA investigators because the assay can be conducted on frozen tissue/cell samples and (b) new assays are readily designed and validated for any protein from any species that has its genome sequenced, e.g., rodents (mice and rats) and humans as well as invertebrates, e.g., yeast, Drosophila, and C. elegans. We envision that this Core will be widely used, e.g., 11 VA investigators at the Oklahoma City VA Medical Center and 10 investigators at VAs across the United States have provided letters expressing their interest in using the Multiplexing Protein Quantification Core. A management team with experience in mass spectrometry and Core management is in place to oversee the Multiplexing Protein Quantitation Core. In addition, a fee-for-service schedule has been developed to pay for the technical support, supplies, and the operation of mass spectrometer in the Multiplexing Protein Quantification Core.
StatusFinished
Effective start/end date1/1/159/30/15

Funding

  • National Institutes of Health

ASJC

  • Medicine(all)

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