Identifying Ciliary Proteins in Mammalian Retinas using a Gentle Extraction Method

Abstract

Mutations in retinal primary cilia are responsible for human blindness but the mechanisms are not fully understood (Wheway et al., 2014). Characterizing the proteome of an organelle such as cilia, is a fruitful way to understand its function but methods often require considerable sample quantities. Here we develop a method to isolate the primary cilia of photoreceptor cells from bovine retinas. Through LC/MS/MS proteomics analysis we identify proteins enriched for cilia function including ciliopathy disease. This study shows our method can be used to isolate retinal primary cilia to obtain sufficient quantities of native protein samples.

Figure 1. Isolation and proteomic analysis of primary cilia from bovine retinas

( A ) A schematic of the methods used to conduct this experiment. The bovine eye was dissected using scalpels and forceps. Next, the rod outer segment of the primary cilia was separated from the retinal tissue using centrifugation. The proteins were extracted and then identified using mass spectrometry (LC/MS/MS). ( B ) From our MS analysis we identified ~1200 bovine proteins; we cross referenced the proteins in our sample with the Syscilia database gold standard and found 23 proteins associated with cilia in our sample. The graph shows the abundance of 23 ciliary proteins within replicate samples. Pink arrow=RP associated proteins and purple arrow=Bardet-Biedl Syndrome associated protein. ( C ) Comparison of our MS bovine protein abundance data compared to proteins identified in mouse retinas (Liu et al., 2007). ( D ) Annotation enrichment analysis of the 40 most abundant proteins in our sample and select significant annotations. Proteins were analyzed by filtering the top 40 proteins by total PSMs across both replicates. (REAC=Reactome, GOCC=Gene Ontology Cellular Component, GOMF=Gene Ontology Molecular Function, PSM = peptide spectral match) (Images in Figure 1A were created with BioRender).