Initiating ocular proteomics for cataloging bovine retinal proteins: Microanalytical techniques permit the identification of proteins derived from a novel photoreceptor preparation

Though some mechanisms of photoreception have been well characterized, othe rs remain obscure. Presumably most, if not all, of the major players in pho toreceptor-specific functions are present in large amounts in the photorece ptor layer, and a catalog of these proteins will prove a useful tool for vi sion researchers. As a first step toward a complete catalog of photorecepto r cells, we have developed a novel method for isolating the photoreceptor c ell monolayer from bovine retina. Electron microscopic studies of both the photoreceptor layer and the residual retina from which the photoreceptor la yer had been removed, indicate that the preparation contains the photorecep tor outer segments and the majority of the inner segments. Proteins were ex tracted from the isolated photoreceptor cell layer as well as the rest of t he retina with isoelectric focusing lysis buffer, and the protein component s were separated by two-dimensional gel electrophoresis. The obtained prote in maps reveal several classes of proteins that appear to be expressed more abundantly or specifically in the photoreceptor layer than in the rest of the retina. Four of these protein spots were excised and in-gel digested wi th trypsin, and the digests were extracted with solvent. The mixture of pep tides digested from each protein was analyzed by high performance liquid ch romatography interfaced with electrospray ionization tandem quadrupole mass spectrometry or by matrix-assisted laser desorption ionization time-of-fli ght mass spectrometry. Some of the peptides were isolated and their sequenc es were determined by gas phase Edman degradation. RNA transcripts extracte d from the photoreceptor layer or the whole retina were subjected to Northe rn blot analysis as well as to reverse transcriptase-polymerase chain react ion amplification of probes for the successful selection of cDNA clones. Th ese data permit both the identification of virtually any protein detectable on a two-dimensional gel, and also enable the corresponding cDNA clone to be selected. We have validated this approach by identifying aspartate amino transferase and creatine kinase from the populations of abundant photorecep tor layer proteins. Both aspartate aminotransferase and creatine kinase are of mitochondrial origin and are thought to play crucial roles in photorece ptor functions by producing glutamate and ATP, respectively. We also identi fied two photoreceptor layer specific proteins: an acidic and high molecula r weight protein, interphotoreceptor retinoid-binding protein, and an acidi c and small molecular weight protein, recoverin. The technique presented here will allow vision researchers to discover and identify the proteins that are expressed specifically or abundantly in the photoreceptor cell as well as the proteins that undergo post-translational modification or modulation in expression under a defined biological conditi on. With the use of this technology, we anticipate that a researcher who kn ows only the 2-D gel position of a protein of interest can identify the pro tein, isolate a cDNA clone, and move into molecular genetic studies. Moreov er, this streamlined technology will enable one to assemble a catalog of ph otoreceptor proteins using a minute amount of materials in a short period o f time. We believe that such a catalog will serve as a valuable resource fo r Vision investigators and will accelerate the rate of research progress. ( C) 1999 Academic Press.