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Nikolai Skiba, Vadim Arshavsky; Complete protein composition of the photoreceptor disc identified by label-free quantitative proteomics. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4079. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Decades of biochemical studies suggested that photoreceptor discs contain a rather small set of structural and signaling proteins. Yet, several proteomic studies identified hundreds of proteins associated with biochemical disc preparations. We addressed whether protein composition of photoreceptor discs is indeed more complex than traditionally believed, or the high sensitivity of mass spectrometry results in identification of many traces of contaminating proteins.
We used quantitative label-free mass spectrometry, a methodology comparing relative amounts of proteins in photoreceptor discs taken from three sequential steps of their biochemical purification from bovine retinas. We expected that constitutive disc proteins should retain a constant molar ratio in all purification steps, whereas the amounts of impurities should gradually decrease as the purity of disc membranes increases.
We have identified 335 proteins in the most pure disc preparation and compared their amounts throughout all three disc preparations. This profiling revealed a small group of 11 proteins whose molar ratios remained constant in all preparations. In contract, the ratios for other proteins decreased as predicted for the impurities. On the top of the latter group, were several proteins, either peripherally associated with the disc membranes or present both in discs and other membranous organelles in photoreceptors. Among the 11 disc-resident proteins, 6 represented well-known components of the phototransduction cascade, 2 - disc-specific lipid flippases ABCA4 and ATP8A2, and 2 - structural proteins peripherin and rom-1. The last protein was PRCD (Progressive Rod/Cone Degeneration). Genetic studies linked mutations in PRCD to severe retinal degeneration in humans and dogs; however, its subcellular localization and function remained unknown.
Our data demonstrate that photoreceptor discs contain only a small group of unique membrane-associated proteins. It further emphasizes the significance of biochemical purity of biological samples in accurate interpretation of proteomics results. The strategy developed and validated in this study can be ubiquitously employed for distinguishing between constitutive and contaminating components in preparations of other cellular organelles and for identification of their novel resident protein constituents.
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