Inherited retinal degenerations (IRDs) are a leading cause of blindness among the young population in the developed world. Approximately half of IRDs initially manifest as gradual loss of night vision and visual fields, characteristic of retinitis pigmentosa (RP). Due to challenges in genetic testing, and the large heterogeneity of mutations underlying RP, targeted gene therapies are an impractical largescale solution in the foreseeable future. For this reason, identifying common key pathophysiological pathways in IRDs that could be used as targets for mutation-agnostic and disease-modifying therapies (DMTs) is warranted. In this study, we investigated retinal proteome of three distinct IRD mouse models, comparing to sex- and age-matched wild-type mice. Specifically, we used the Pde6βRd10 (rd10) and RhoP23H/WT (P23H) mouse models of autosomal recessive and autosomal dominant RP, respectively, as well as the Rpe65-/- mouse model of Leber´s congenital amaurosis type 2 (LCA2). The mice were housed at two distinct institutions and analyzed using LC-MS in three separate facilities/instruments following data-dependent and data-independent acquisition modes. This cross-institutional and multi-methodological approach signifies the reliability and reproducibility of the results. The largescale retinal proteome profiling, coupled with in vivo electroretinography recordings, provided us with a reliable basis for comparing the disease phenotypes and severity.

The mouse retinas were homogenized in 100 µl of protein extraction buffer (ab193970) (Abcam, UK) using a handheld homogenizer (Pellet Pestle Cordless Motor, FisherScientific) for 30 s on ice. The retina homogenates were then solubilized by sonication in two 30-s bursts, interspersed with 30-s intervals of rest on ice. The lysates were then centrifuged at 18,000 g for 20 min at 4 ºC. The supernatants (extracted solubilized proteins) were collected in separate Eppendorf low protein binding tubes (Thermo Fisher Scientific, MA, USA). Total protein content was measured from retinal lysates using BCA pierce protein assay (Thermo Fisher Scientific, MA, USA), and the total protein concentration was adjusted to 1 mg/ml for all samples using the same lysis buffer. The extracted and solubilized retinal proteins were processed using FASP, as previously described (Montaser et al., 2022; Wiśniewski, 2019). Proteomics analysis was conducted using an UPLC (Vanquish Flex, Thermo Scientific, Bremen, Germany) coupled to a high-resolution Orbitrap Q Exactive Classic mass spectrometer (Thermo Scientific, Bremen, Germany) operating in positive ion mode. Mobile phase A consisted of 0.1 % FA in water, while mobile phase B comprised 0.1 % FA in ACN. Peptides were loaded into an Agilent AdvanceBio Peptide Map column (2.1 mm × 250 mm, 2.7 μm, Agilent Technologies, Santa Clara, CA, USA). The flow rate was set at 0.3 mL/min, and peptides were separated over an 80-min active gradient from 2 % to 45 % buffer B (total method duration 90 min). Mass spectrometric detection encompassed Full MS–SIM (Resolution: 35k; AGC target: 3e6; max injection time: 60 ms; scan range: 385–1015 m/z) and DIA (Resolution: 17.5k; AGC target: 2e6; max injection time: 60 ms; loop count: 25; isolation window: 24 m/z).

UEF_DIA_rd10_DR_cohort 3: rd10 (N=4; n=2 females, n=2 males) and WT (N=4; n=2 females, n=2 males) were housed in a dim light environment at UEF-LAC throughout their lifespan. The mice were kept in a Scantainer®, in which glass doors were covered with a darkening film, limiting light exposure to < 0.01 lux inside the mouse cages. The rd10 and WT mice were euthanized in the same session with a 3 min exposure to CO2 and subsequent cervical dislocation at P23 and P25, respectively.