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A mouse photoreceptor proteome resource

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Retinal Rod Cell, Retinal Cone Cell, Retina

SUBMITTER: Olaf Jahn

LAB HEAD: Prof. Dr. Olaf Jahn

PROVIDER: PXD066755 | Pride | 2025-12-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2024_03_Sprot_17212_retina_SNAP25_PALS2_TRYP_ENO_EcoliHi3_reverse.fas	Other
	210811_S2_FASP_210809_Retina-Erlangen_1830-1_HDMSe_9ul-01.raw.zip	Raw
	210811_S2_FASP_210809_Retina-Erlangen_1830-1_HDMSe_9ul-01_IA_final_peptide.csv	Csv
	210811_S2_FASP_210809_Retina-Erlangen_1830-1_HDMSe_9ul-01_IA_final_protein.csv	Csv
	210811_S2_FASP_210809_Retina-Erlangen_1830-1_HDMSe_9ul-02.raw.zip	Raw

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Project description:Rod and cone photoreceptors in mammalian retina are generated from common pool(s) of neuroepithelial progenitors. NRL, CRX and NR2E3 are key transcriptional regulators that control photoreceptor differentiation. Mutations in NR2E3, a rod-specific orphan nuclear receptor, lead to loss of rods, increased density of S-cones, and supernormal S-cone-mediated vision in humans. To better understand its in vivo function, NR2E3 was expressed ectopically in the Nrl-/- retina, where post-mitotic precursors fated to be rods develop into functional S-cones similar to the human NR2E3 disease. Expression of NR2E3 in the Nrl-/- retina completely suppressed cone differentiation and resulted in morphologically rod-like photoreceptors, which were not functional. Gene profiling of FACS-purified photoreceptors confirmed the role of NR2E3 as a strong suppressor of cone genes and an activator of a subset of rod genes (including rhodopsin) in vivo. Ectopic expression of NR2E3 in cone precursors and differentiating S-cones of wild type retina also generates rod-like cells. The dual regulatory function of NR2E3 is not dependent upon the presence of NRL and/or CRX, but on the timing and level of its expression. Our studies reveal a critical role of NR2E3 in establishing functional specificity of post-mitotic photoreceptor precursors during retinal neurogenesis. Experiment Overall Design: We mated the Crx::Nr2e3/Nrlko mice with the Nrl::GFP transgenic mice, in which the expression of GFP is driven by an Nrl promoter. Mouse retinas were dissected at 4 wk. GFP+ photoreceptors were enriched by FACS (FACSAria, BD Biosciences, Franklin Lakes, NJ). RNA was extracted from 1~5x105 flow-sorted cells using Trizol (Invitrogen). Total RNA (40-60 ng) was used for linear amplification with Ovation Biotin labeling system (Nugen), and 2.75 ug of biotin-labeled fragmented cDNA was hybridized to mouse GeneChips MOE430.2.0 (Affymetrix) having 45,101 probesets (corresponding to over 39,000 transcripts, and 34,000 annotated mouse genes). Experiment Overall Design: Four independent samples were used at 4 weeks. We normalized Experiment Overall Design: Crx::Nr2e3/Nrl-ko-Gfp data along with Nrl-ko-Gfp 4 weeks samples ( 4 replicates, refer to Series submission GSE4051). The normalized data was then subjected to two stage analysis based on False Discovery Rate Confidence Interval (FDR-CI) for screening differentially expressed genes (24, 27) with a minimum fold change of 4.

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A mouse photoreceptor proteome resource

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