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M. E. Algeciras, D. Ding, S. K. Bhattacharya; Determination of Optimal Methods for Detection of Retinal Deimination. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3407.
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Deimination is a recently discovered long term posttranslational conversion of protein bound arginines into citrulline, prevalent in the retina and the optic nerve. The objective of our investigation is to determine optimal conditions for identification and capture of retinal deiminated proteins and the sites of deimination within the proteins.
Human, pig and rat (n= 5) retinal extracts and recombinant CRALBP, BSA and purified myelin basic protein (MBP) were used for these studies. The protein mixtures were subjected to monoxime treatment using 2, 3 butanedione and antipyrene in acidic environment. A series of experiments were performed using in-gel or in solution monoxime treatment followed by separation on SDS-PAGE or on TLC plates. Mass spectrometry of synthetic peptides and chymotrypsin digested peptides were performed to identify the sites of deimination.
The deiminated proteins underwent aggregation as a function of period of monoxime treatment in-solution. Monoxime treatment in-gel prevented aggregate formation but resulted in cross-linking with polymer matrix. In-gel but not in-membrane (PVDF) monoxime treatment enabled partial capture of modified deiminated proteins. Monoxime treatment on TLC or separation of treated peptides on TLC enabled better capture and identification of deiminated peptides.
These investigations provided optimal conditions for better capture of deiminated proteins and peptides. The separation of peptides or treatment of proteins immobilized on TLC resulted in identification of deimination sites on peptides. Such separation also enabled better identification of deiminated proteins from a complex mixture of proteins. The new and improved method will allow better capture and identification of deiminated proteins in the retina and optic nerve.
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