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J.S. Crabb, X. Gu, M. Nawrot, J.C. Saari, J.W. Crabb; Quantitative Mass Spectrometric Analysis of CRALBP–Protein Interactions . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2039.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies suggest that cellular retinaldehyde–binding protein (CRALBP) functions in the rod visual cycle as a component of a RPE retinoid– processing protein complex. Mass spectrometric methods are under development for the identification and relative quantification of CRALBP–protein interactions.
CRALBP–protein interactions were sought in bovine RPE microsomes by immunoprecipitation with CRALBP antibody covalently bound to agarose beads. Immunoprecipitated proteins were digested with trypsin, peptides labeled with iTRAQ amine–specific isobaric tags and identified and quantified by LC MS/MS. IP products were fractionated either by SDS–PAGE prior to tryptic digestion or by SCX ion exchange chromatography after proteolysis. Quantification was obtained relative to control samples of RPE microsomes.
CRALBP, RPE65, LRAT, RDH5, and RGR opsin co–precipitate from RPE microsomes with anti–CRALBP antibodies along with a variable number of other proteins. Other proteins include photoreceptor and blood components as well as other potential RPE visual cycle components. Quantification of experimental and control samples is in progress to provide discrimination between background contaminants and authentic, CRALBP interaction partners.
Visual cycle proteins CRALBP, RPE65, LRAT, RDH5, and RGR opsin may interact in a RPE protein complex; however, further analyses are required to confirm this possibility. Toward this end, comparative quantification of visual cycle reciprocal immunoprecipitatation products and of nonspecific interactions with the affinity support may be useful.
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