Multiplex proteomic analysis by two‐dimensional differential in‐gel electrophoresis

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Abstract
This paper describes the use of fluorescence two‐dimensional differential in‐gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK1R−/−) and wild type (NK1R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK1R−/− and NK1R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two‐dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In‐gel Analysis software. A Cy2‐labeled sample pool was co‐electrophoresed with all Cy3‐ and Cy5‐labeled sample pairs as an internal standard providing a link for inter‐gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK1R−/− mice compared to NK1R+/+ controls. Matrix assisted laser desorption/ionisation‐time of flight (MALDI‐TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.