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N. P. Skiba, E. S. Lobanova, V. Y. Arshavsky; Mapping Proteins in Subcellular Compartments of Rod Photoreceptors Using Mass Spectrometry Approach. Invest. Ophthalmol. Vis. Sci. 2007;48(13):594.
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© ARVO (1962-2015); The Authors (2016-present)
To generate a proteome map of the rod photoreceptor cell where a large number of individual proteins will be assigned to their relative subcellular locations.
We first obtained 10 micrometer serial tangential sections through the photoreceptor layer of the frozen flat mounted rat retina. The intracellular origin of each section was determined by Western blot analysis of specific intracellular markers. Sections representing the major intracellular compartments of the rod, outer segments, inner segments, stacks of nuclei and synaptic terminals, were analyzed using the 1D SDS-PAGE-LC/MALDI workflow.
At the first phase of this study we analyzed protein contents in two combined sections representing each individual subcellular compartment and identified over 350 proteins. Among them 39 proteins were unique to the outer segment. This included 11 well-characterized phototransduction proteins. 38 proteins were unique to the inner segment, 32 to the nuclear layer and 45 to the synaptic terminal. 24 proteins were found in all four sub-cellular compartments. For every previously characterized protein, its distribution corresponded to the published subcellular localization. More than 30 proteins identified in this experiment are not annotated in the NCBInr protein database and predicted based on the DNA sequencing data. It is likely that many of them will be confirmed to be novel, not previously characterized proteins. We are now conducting the second "large scale" stage of this study where the amount of initial material is at least 10-fold larger than in the first experiment.
Our data indicate that a combination of serial cryosectioning of the photoreceptor cell layer with mass spectrometry provides a wealth of information on protein localization in their sub-cellular compartments. The sensitivity of this approach is quite remarkable and the total number of identified proteins is expected to be significantly increased by combining multiple sections representing the same subcellular compartment for the mass spec analysis.
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