Isolating Photoreceptor Compartment-Specific Protein Complexes for Subsequent Proteomic Analysis

Abstract

Tulp1 is a photoreceptor-specific protein that may perform two distinct functions, one in the inner segment (IS) and another in the synapse. To differentiate the roles of Tulp1 in the photoreceptor, we developed a methodology for the capture of compartment-specific complexes for subsequent protein analysis. Using in vivo perfusion, we crosslinked proteins to conserve endogenous protein complexes. Laser microdissection was used to collect three compartment tissue samples from retinal sections: the IS, the outer plexiform layer (OPL) containing the photoreceptor synapses, and the inner plexiform layer (IPL), serving as a Tulp1-free control tissue. Compartment-specific as well as whole retinal samples were homogenized and subjected to Western blot analysis. Our analysis showed that a band at approximately 78 kDa labels native Tulp1 in the wt crosslinked tissue, but not tulp1 ^−/− crosslinked tissue. Two additional bands were detected at ∼180 and ∼280 kDa in wt homogenate, indicating the presence of Tulp1 complexes. Finally, a band matching the 280 kDa band was present in the IS-isolated sample, but not the OPL-isolated sample, indicating an IS compartment-specific Tulp1 complex.