Absolute quantification of photoreceptor outer segment proteins.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously measure the outer segment content of twenty key structural and functional proteins. We determined the molar ratio amongst all twenty proteins as well as the number of molecules of each protein residing within an outer segment. To assess the precision of this quantification, we took advantage of the fact that seven of these proteins exist within three constitutive complexes of well-established subunit stoichiometries. Remarkably, our measurements differed from these stoichiometries by less than 7%, highlighting the exceptional precision of our quantification. This allowed us to resolve the existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.