Profiling of the global proteome and the repertoire of N-termini of vitreous humor uncovers alterations of cell adhesiveness and extracellular matrix composition in rhegmatogenous retinal detachment

Rhegmatogenous retinal detachment (RRD) is a severe eye condition characterized by the detachment of photoreceptors layer, and of the whole retina indeed, from the retinal pigment epithelium (RPE). The disease carries a heavy burden for the quality of life and for social and health costs, as a significant fraction of subjects experiences a second detachment few months after the first episode, this occurrence being associated with sight loss.

Under healthy conditions, the adhesiveness between the two layers is mainly promoted by the mechanical compression exerted by the vitreous humor (VH), a fluid which shapes the eye globe and nourishes the tissues. Hence, alterations of VH composition and of the extracellular matrix remodeling are thought to underscore the disease onset, but the molecular mechanisms are largely unknown yet.

Based on these premises, profiling of the perturbations of the VH proteome, may provide clues for deciphering the early molecular events triggering the RRD, thereby helping the clinicians in predicting the fraction of subjects who are more vulnerable to a second detachment.

Therefore, we have here undertaken a shot-gun/bottom-up proteomic characterization of the VH isolated from primary RRD subjects (n=9) and subjects diagnosed with macular holes or pucker (controls, n=9), which are two pathologies not classifiable as retinal detachment but which are treated by VH surgery as well. Age and gender ratio were comparable between the two experimental groups as well as the general inclusion and exclusion criteria.

Tryptic digests were then injected into a Orbitrap Exploris 240 (2 injections for each individual sample) coupled with a nano-HPLC system. Raw data were searched against a human proteome for exploring the composition of global VH proteome (Sequest enabling Inferys rescoring, in Proteome Discoverer 2.5) and the repertoire of natural endogenous N-termini (semi-tryptic search using FragPipe).

By setting FDR<0.01 (target-decoy) and filters for identification of unique proteins, 869 proteins were identified in the VH, and up to 721 N-termini were identified.

After having applied a quantile normalization, and verified quality controls (e.g., data distribution, etc.), analysis of differentially expressed proteins and N-termini, followed by data clustering and rationalization, revealed a significant perturbation of key pathways for cell adhesiveness (Wnt signalling), matrix components, stress responses and immune responses.

Data obtained suggest that RRD is effectively sustained by global alterations of the VH proteome and that aberrant proteolytic events on extracellular matrix components may serve roles for disease onset and progression.