A Modified Acyl-RAC Method of Isolating Retinal Palmitoyl Proteome for Subsequent Detection through LC-MS/MS

Background

The retina is a thin layer of neural tissue lining the back of the eye, containing six different cell types organized together for visual perception. Vision is initiated through retinal photoreceptors, which are polarized and compartmentalized neurons containing ciliated outer segments (OS). Photoreceptor OS contains thousands of double membranous discs that harbor phototransduction proteins, which are responsible for capturing light and converting it into an electrical signal. The OS discs, along with the proteins that mediate phototransduction within the ciliated structures, are phagocytized in a circadian rhythm by pigmented epithelial cells that abut the retinal layer. On the basal side of the photoreceptor neurons lay the ribbon synapses that are responsible for the transmission of light-dependent electrical signals to downstream neurons, which ultimately transmit the signals to the visual cortex. The proteins present at the synapse and in the OS disc membranes are synthesized in the inner segment (IS) of the photoreceptor neurons and trafficked to their destination. The constant renewal of the OS discs necessitates continual protein synthesis and efficient sorting machinery (Burns and Arshavsky, 2005; Baker et al., 2008).

The protein trafficking machinery in photoreceptor neurons is greatly aided by the lipid modification of proteins (Kolandaivelu et al., 2009;Maeda et al., 2010;Yang and Wensels, 1992). Among the lipid modifications, palmitoylation is the most common form of protein acylation, which adds a 16-carbon palmitic acid to a cysteine residue in the protein. Unlike most lipid modifications, palmitoylation is unique and reversible, and therefore plays a dynamic role in dictating the localization of a protein both spatially and temporally. Defects in the sorting of proteins due to the absence/loss of palmitoylation have been associated with various inherited blinding diseases, suggesting an important role of this lipid modification. Several studies including ours described the importance of palmitoylation in retinal health and function. We showed that lack of palmitoylation in a retinitis pigmentosa–associated protein—progressive rod-cone degeneration (PRCD)—leads to protein mistargeting to the photoreceptor IS where it undergoes severe destabilization (Murphy and Kolandaivelu, 2016). Likewise, mutating palmitoylation sites in rhodopsin leads to severe light-induced photoreceptor degeneration (Maeda et al., 2010). However, a paucity of information on retinal palmitoylated proteins hinders our progress toward understanding the significance of this crucial modification in retinal structure and function. Our modified acyl resin–assisted capture (Acyl-RAC) method efficiently identifies the global palmitoyl proteome in retinal neurons and other tissues that are compatible with downstream mass spectrometry (LC-MS/MS) analyses for further validation (Forrester et al., 2011;Murphy and Kolandaivelu, 2016; Myers et al., 2022). Using our protocol, we show the efficient isolation of palmitoylated proteins and downstream validation of known palmitoylated proteins by immunoblotting. Using this protocol, we show that several proteins that are critical for proper retinal function, and proteins linked with blinding diseases, are post-translationally lipid-modified by palmitoylation. Additionally, we show that our protocol can be reliably used to identify palmitoylated proteins in other tissues.