This proposal is for the acquisition of a high-end LC-MS/MS instrument to be placed in the Proteomics Core Laboratory at the Lerner Research Institute at the Cleveland Clinic. The proposal contains projects from twelve NIH funded investigators and is asking for a Thermoscientific LTQ-Orbitrap Velos instrument equipped with an Eksigent nano1D plus HPLC. This is a high-end LC-MS/MS system that represents the state-of-the-art in mass spectrometry that is being utilized for proteomics investigations. Currently, the lab is equipped with two LC-MS/MS systems including a ten year old Thermoscientific LCQ deca and seven year old Thermoscientific LTQ instrument. These instruments have been the cornerstone of the Proteomics Core, which for the past ten years has been a productive lab providing high quality work to investigators not only at the Lerner Research Institute but the greater Cleveland Scientific community. These instruments can no longer keep up with the current requests for global proteomic quantitative experiments and post-translational modification studies from investigators at the Lerner Research Institute. We have performed a direct comparison of the LTQ and LTQ-Orbitrap Velos instruments and observed an increase by a factor of 3 to 4 in the total number of proteins identified, an increase in mass accuracy of over an order of magnitude (5 ppm vs 150 ppm), and an increase in resolution of almost an order of magnitude (50000 vs. 6000) for the LTQ-Orbitrap Velos instrument compared to the LTQ. The acquisition of this state-of-the-art instrument will allow the Proteomics Core to more completely analyze any given proteome, enhance the ability to identify low abundant proteins or post-translational modifications, increase the accuracy of label free quantitation methods, more confidently identify post-translational modifications, and allow the researchers at the Lerner Research Institute to start utilizing stable isotope labeling in quantitative experiments. This instrument will be applied to a variety of projects including the identification of substrates for the protease ADAMTS5 (Apte lab), qualitatively and quantitatively study proteasomal degradation of cytoskeletal proteins in human platelets (McIntrye lab), more completely understand the translational control of inflammatory gene expression by the GAIT complex (Fox lab), elucidate the protein expression changes induced by mislocalization of PTEN in the context of breast carcinogenesis (Eng lab), perform a quantitative proteomic analysis of ethanol and homocysteine induce hepatic cell injury (Jacobsen lab), determine the mechanism of topo IIb regulation of retinoic acid-induced differentiation of myeloid cell (Ganapathi lab), and phosphoproteomic analysis of vascular smooth muscle cell response to antiotensin II (Karnik lab).
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