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 Center for the Analysis of Cellular Mechanisms has establish a New Proteomics Reagent Synthesis Core. In the process of developing the new Zdye multiplex fluorescent protein dyes for detecting changes in protein levels after biological stimuli, Prof. Grieco has developed considerable expertise in fluorescent dye synthesis and preparing variations of the dyes targeted for various functional groups. Professor Grieco will direct all aspects of this Core, which will provide new multicolor fluorescent proteomic reagents based on the new Zdye fluorphore family. New differential Activity-Based Protein Profiling (dABPP) reagents are presented in this Core Description and differential Thiol Redox Protein Analysis (dThiRPa) reagents also produced by this Core will be characterized in the Singel COBRE Project (Project-3). Projects 1, 2 and 4 will make substantial use of these new dABPP and dThiRPA reagents. The ABPP field is expanding rapidly to identify new reactive groups for identifying changes in new classes of enzyme activities. Trifunctional capture reagents will be developed for all of the enzyme activity classes that will be used to capture the labeled proteins for identification of their active site regions. These unique differential reagents will be made available to all users of the Center whether they have COBRE projects or not. In addition, there is a need for additional colors of Zdyes so that differential analysis of more variables can be carried out simultaneously. Thus, we expect that this Core will remain busy throughout the entire COBRE project period, in the later periods making reagents that have not been designed as yet. The basic technology entails covalently labeling protein fractions from cells treated in various ways with different-colored fluorescent dyes with suitable reactivity, mixing the labeled proteins and separating them on 2D gels, as described in the main body of the proposal. Laser scanning reveals which proteins have changed due to the different biological treatments of each sample. The 2D gel approach has the significant advantage, compared to other proteomics technologies, that most protein post-translational modifications shift the modified form to a new position on the gel (Halligan. Ruotti et al. 2004;Kumar. Khachane et al. 2004). From the perspective of differential analysis, this is a best case scenario because the appearance of a new spot is relatively easy to detect. Protein post-translational modifications control a very large fraction of biological mechanisms, however their detection has proven to be problematic for the alternative proteomics methodologies, particularly when monitoring global changes in protein post-translational modifications. The dABPP and dthiRP approaches outlined in this COBRE proposal take advantage of the strength of 2D gel methods to provide a global map of the relative amounts of each protein form and will allow the capture of the proteins of interest for identification in the mass spectrometer and active-site identification by detection of the labeled sites. There have been many well-recognized limitations to 2D gel technology and efforts have been mounted at MSU to combine the best available technology and to add improvements to overcome the past limitations in 2D gel technology for global differential proteomic analysis. Competing commercial GE DIGE dyes have previously provided a major improvement by overcoming problems of gel-gel reproducibility, but have limited sensitivity and labeling with DIGE dyes can reduce the recovery of labeled proteins on the gel patterns. The new Zdyes are designed to be highly water soluble to enhance the solubility of the labeled proteins at their isoelectric points, so that more protein can be loaded for more sensitive analysis and the high Zdye water solubility tends to enhance recovery of labeled proteins and transfer from the first to the second dimension in 2DE.
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