A mouse photoreceptor proteome resource

To enrich mouse rod and cone photoreceptors for mass spectrometric analysis, we optimized our previously established trituration-based fluorescence-activated cell sorting (FACS) strategy (see Lux et al., 2024 for details; PMID: 38481472). For quantitative mass spectrometry, we utilized an ion mobility-enhanced version of a data-independent acquisition (DIA) workflow with alternating low and elevated energy (referred to as UDMSE). Label-free protein quantification of rod and cone photoreceptor in comparison with retina samples revealed an enrichment of photoreceptor-specific proteins. More importantly, established photoreceptor markers were significantly enriched in the respective photoreceptor samples, which confirms the quality and specificity of our proteome resource. Among the proteins highly enriched in the cone photoreceptor samples, we identified Mpp6/Pals2, a protein uncharacterized in photoreceptors so far, and established it as a novel pan-cone photoreceptor marker by immunocytochemistry. As an example of the use of our resource for the characterization of retina- and photoreceptor-specific protein isoforms, we assessed the expression of the two known isoforms of Mpp6/Pals2, the canonical Pals2β and the 14 aa shorter splice variant Pals2α. We confidently identified a tryptic peptide that only occurs upon splicing, indicating the expression of the non-canonical Pals2α in cone photoreceptors. In summary, our proteome resource provides comprehensive details on the protein (isoform) composition of mouse rod and cone photoreceptors, making it a valuable research tool for the study of retina biology and pathology.