Abstract
Purpose :
Retinal photoreceptors are neurons generating electrical signals in response to light excitation. This function is performed by a set of phototransduction proteins localized in photoreceptor outer segments. Despite their well-known functions, the information about their absolute amounts is often conflicting, which impedes our deeper understanding of the relevant molecular mechanisms. In this study, we used a recently developed mass spectrometry-based protein quantification method, called “MS-Western”, for measuring absolute amounts of key functional proteins residing in rod outer segments.
Methods :
MS Western quantifies proteins after co-digestion of a sample containing proteins of interest combined with a heavy-isotope labeled protein chimera composed of tryptic peptides representing each of these proteins and a reference protein (typically BSA). It relates the molar abundance of all analyzed peptides to a single reference protein (BSA) added to the sample as a standard. Our chimeric construct contained 3-5 reliably identifiable tryptic peptides representing 20 rod outer segment proteins along with 6 peptides representing BSA.
Results :
By comparing the ion intensities of “light” and “heavy” peptides measured in a single mass spectrometry experiment, we simultaneously measured the absolute amounts of all 20 proteins of interest. The method’s accuracy was validated by confirming the equimolar amounts of protein subunits within the known stoichiometric complexes: PDE6α/PDE6β and RGS9/Gβ5/R9AP. The ratios of PDE holoenzyme and the RGS complex to rhodopsin in mouse rods were 1:120 and 1:450, respectively. Another example of this quantification is the 6:1 molar ratio between guanylyl cyclase 1 and 2, and their respective molar ratios to rhodopsin of 1:510 and 1:2,970. Altogether, this analysis provided an accurate quantification of 20 phototransduction and other functionally important outer segment proteins and resolved several discrepancies on their quantifications previously reported in the literature.
Conclusions :
MS-Western is an accurate method for quantification of multiple proteins in a single mass spectrometry experiment. This approach is by far superior to a traditional Western blotting technique, which requires high quality antibodies and purified protein standards. MS-Western can be used to accurately measure the amounts of multiple proteins in any ocular tissue
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.