This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Director of the Mass Spectrometry and Proteomics Core oversees the Mass Spectrometry and Proteomics Core, the staffing, the Short Course Training Program, New User Hands-On Training Program, oversee regular monitoring of instrument performance, and leads, with the assistance of the PI, discussions of new directions in instrumentation and technology that should be considered and tested. Training of new users in MALDI, DeCyder, Progenesis, Mascot, and SpectraMill will be carried out. Staff will include two full time technicians, one supported by COBRE and the second by University and Facility funds, and a 3/4 time instrument maker. The Director will interact with the equipment vendors to report performance issues, arrange for board-swap repairs to be made, vendor site visits, and software upgrades. He assists the PI in preparing progress reports, grant renewals and new grant proposals for infrastructure enhancements that could enhance research project performance, but were not anticipated by the present proposal. The Research Technician will be responsible for training new users in 2D gel technology, in protein digestion and the operation of the nanoLC/ChipLC/lon Trap LC/MS/MS. He/she will carry out limited service work using these technologies, in cases were laboratories are motivated to try their samples and their research problems with the new proteomics infrastructure and technology, but do not have the personnel available to learn the technology. If initial experiments are successful the laboratories will be expected to put someone forward to learn to carry out and interpret the experiments. He/she will also be responsible for running and recording frequent quality control tests. Instrument Maker (75% time) will build, test and maintain a targeted gel spot electro-elution device mounted on a gel spot picking robot and will coordinate the building and testing of a serial IEF apparatus. The new Zdye proteomics multiplex detection system can pin-point protein spots that change with biological stimulation (for protein amount, protein activity, and/or in selected post-translational modifications). The in-gel digestion and peptide extraction approach currently used to obtain samples for mass spectrometric analysis tends to lose lower abundance samples, due to poor digestion and losses on wells, pipet tips and tubes. The multicolor/multiplex gel scanning optimizes the location of protein spots from cells or tissues that change with biological stimulation. The device under construction will electro-elute proteins from the targeted spots, flow the sample through a microfluidic immobilized trypsin digester, and collect the sample in a multiplate well for MS analysis or onto a Maldi plate for initial testing. We plan to ultimately flow the eluted peptides directly into the trapping column of the Agilent Chip LC, for the most efficient and high-recovery analysis. The electro elution path is short (the 1.0-1.5mm gel thickness) and the immobilized trypsin digester is also fast and each targeted spot will take about 10 min to elute and digest. A vast number of spots are resolved in typical experiments but only a relatively small number of protein spots show sufficiently large changes in intensity, with biological stimulation to require identification. The stronger spots can be removed and processed by the standard spot-cutting robot protocol in use or by electro-elution and microfluidic digestion. Many of the """"""""control"""""""" proteins in cells, such as transcription factors, are present in low copy number and their active forms are typically a minor fraction that is phosphorylated. Modifications, such as phosphorylation, shift the proteins to new positions on 2D gels and so changes in the intensities of weak protein spots often contain a great deal of biological information. The proteins that change in the weaker spots will be recovered by the electro elution device to be constructed. The stronger spots that change with biological stimulation will be recovered by the standard spot cutting robot system in use now. The HyperScan system will provide much faster scanning (one pass for all colors and measurement of about 400 pixels per pass) than conventional laser scanners that must carry out independent scans for each single laser excitation and emission filter combination. The current best laser gel scanner is the GE Typhoon Trio (which we have at MSU) that takes 20 min/per scan color on large format gel, and a minimum three color scans per gel, for a long total scan time if multiple gels are used per experiment. The HyperScan will be about 10x faster, be more sensitive, and will also allow expansion to use many more simultaneous multiplex colors (with no decrease in scan time) and allow much more overlap of emisson spectra, as more Zdye colors become available. Four colors are in hand, five colors will soon be available, and eight simultaneous colors are feasible for dose-response curves, time-response curves, simultaneous monitoring protein amounts and post-translational modifications, or comparison of many more samples simultaneously for greatly expanded sample throughput and information output.
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