Funds are requested for a two dimensional difference gel electrophoresis (2D-DIGE) sample preparation system for proteomics, consisting of devices for: separation, fluorescence imaging, computerized data analysis, robotic spot-picking, proteolytic digestion, and delivery to targets suitable for mass spectrometry analysis. This system provides a semi-quantitative assessment of differential protein expression with enhanced reproducibility between pairs of samples, since fluorescent dyes of different emission spectra are utilized to label the samples. As a result, statistical reliability is increased, and precious samples are more efficiently used. This feature is even more critical for proteomics analysis of samples prepared using the cell sorter or of laser captured cell populations, as described in some projects. The imaging device also permits comparison of protein patterns with potentially phosphorylated proteins detected using a fluorescent dye and with immunoreactive proteins. Investigators participating in this proposal have proteomics applications in signal transduction, cell communications, obesity, diabetes and cancer research and biological discovery. This instrument system will also be useful for testing feasibility of new projects not included in this proposal. The instruments will be operated and supervised by experienced protein chemists and mass spectrometrists within an existing shared protein core facility with a record of productivity and collaborative interactions.
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