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M.M. Jablonski, G. McCollum, J.S. Penn, X.F. Wang; Proteomic Analysis of the Retina: Identification of Key Players in Photoreceptor Outer Segment Assembly . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1089.
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To determine which retinal proteins likely play a critical role in photoreceptor outer segment (OS) assembly. This was accomplished by identifying which proteins were preferentially expressed by the retina when OS were properly assembled. The inclusion of positive and negative controls allowed us to rule out proteins whose expression was not necessary or sufficient to support OS assembly.
We analyzed and compared the proteome maps from Xenopus laevis tadpole eyes obtained from 4 experimental conditions: (1) RPE–supported retinas; (2) RPE–deprived retinas; (3) RPE–deprived retinas cultured with IPTG, a glycan that supports OS assembly; and (4) RPE–deprived retinas cultured with mannose, a glycan that does not support OS assembly. After three–days, eyes were harvested. Solubilized proteins were labeled with CyDye fluors followed by multiplexed 2D separation. The inclusion of a pooled standard was utilized to minimize inter–experiment variability. Four experimental replicates were performed, for a total of 16 multiplexed gels. Intensity of protein spots and comparison of proteome maps was performed using DeCyder software.
The comparison of the protein maps from our four experimental conditions is currently in progress. During our evaluation to determine which proteins are differentially regulated under our experimental conditions in which the assembly of OS was modulated, we will make the following assumptions and interpretations. If we determine that a protein spot is equal in quantity in both the proteome map of RPE–supported retina and that from RPE–deprived retinas, we will conclude that that protein is not differentially regulated and not sufficient to support proper OS assembly. In contrast, a protein will be defined as differentially regulated if, compared to RPE–supported retinas, the spot intensity varies by >1.5–fold compared to RPE–deprived retinas, the spot intensity is restored to normal or near normal levels in RPE–deprived eyes exposed to IPTG and it does not return to normal levels in RPE–deprived eyes exposed to mannose. Protein spots meeting the aforementioned criteria for being differentially regulated will be identified using mass spectrometry.
2D–DIGE is a powerful tool for detection of differences in protein expression levels between our 4 experimental conditions. Identification and validation of proteins that are preferentially expressed in retinas with organized OS is the first step in determining the key molecules involved in regulating photoreceptor OS assembly.
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