We have identified several NIH funded Investigators and two multi-institution core facilities whose research projects involve investigation of how changes in protein expression relate to abnormal cellular and/or organismal physiology. Major user projects included in this application cover a variety of disciplines, including: hematology, rheumatology, AIDS research, cardiology, oncology and cell biology. We are requesting an integrated system including a laser scanner (Typhoon Trio), spot cutter (Ettan Spot Picker), driver/analysis software and necessary computing. This integrated system will enable users to perform comparative analysis of protein expression using 2-D gels, to quantify the magnitude of differential expression, and to isolate and identify proteins present in differential 'spots'-the latter made possible by coupling with existing LC-MS capability at TSRI. The major new capability this shared equipment confers is the ability to multiplex protein samples to perform DIGE analysis as described below. DIGE (difference gel analysis) is a more accurate, reproducible and less cumbersome method for comparative proteome analysis of replicate samples. This system will be housed in an existing, NCI funded Proteomics Core located in the Department of Molecular and Experimental Medicine (MEM) at TSRI. Objective: The long-term objective of this proposal is to provide researchers at TSRI and neighboring institutions a reliable, reproducible and quantitative method for comparative proteomic studies.
Specific Aim : Establish Comprehensive DIGE service in the MEM Proteomics Core. We will purchase a Typhoon variable mode imager and Ettan spot picker and integrate this equipment with existing services to provide a comprehensive DIGE capability. Users will determine the extent of core support required for their project. Comprehensive core services will include: labeling protein samples with cyanine fluorophores, 2-D electrophoresis, image capture on Typhoon and data analysis using Decyder software; generation of spot pick list and cutting using Ettan spot picker; and processing of protein spots including digestion, LC-MS and protein database queries of MS profiles. One dedicated computer workstation will be provided for data analysis using Decyder and Decyder extended data analysis software. It will also serve as a training station for new users learning software applications and will have access to online tutorials for analysis software. A second 'floating' license will be maintained for software to allow remote data analysis by groups not located near the facility. Relevance: Identified user groups are all involved in disease specific research that is highly relevant to human health, and receive support from several NIH institutes. Comparative proteomic studies such as those outlined in this application are complementary to gene expression array data, but provide additional insight into how changes in gene activity translate into disease phenotypes by altering protein abundance, protein isoforms and protein modifications. ? ? ?
