To expedite gene discovery in eye development and its associated defects, we previously developed a bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery). However, iSyTE is presently limited to lens tissue and is predominantly based on transcriptomics datasets. Therefore, to extend iSyTE to other eye tissues on the proteome level, we performed high-throughput tandem mass spectrometry (MS/MS) on mouse embryonic day (E)14.5 retina and retinal pigment epithelium combined tissue and identified an average of 3,300 proteins per sample (n=5). High-throughput expression profiling-based gene discovery approaches-involving either transcriptomics or proteomics-pose a key challenge of prioritizing candidates from thousands of RNA/proteins expressed. To address this, we used MS/MS proteome data from mouse whole embryonic body (WB) as a reference dataset and performed comparative analysis-termed "in silico WB-subtraction"-with the retina proteome dataset. In silico WB-subtraction identified 90 high-priority proteins with retina-enriched expression at stringency criteria of ³2.5 average spectral counts, ³2.0 fold-enrichment, False Discovery Rate <0.01. These top candidates represent a pool of retina-enriched proteins, several of which are associated with retinal biology and/or defects (e.g., Aldh1a1, Ank2, Ank3, Dcn, Dync2h1, Egfr, Ephb2, Fbln5, Fbn2, Hras, Igf2bp1, Msi1, Rbp1, Rlbp1, Tenm3, Yap1, etc.), indicating the effectiveness of this approach. Importantly, in silico WB-subtraction also identified several new high-priority candidates with potential regulatory function in retina development. Finally, proteins exhibiting expression or enriched-expression in the retina are made accessible in a user-friendly manner at iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), to allow effective visualization of this information and facilitate eye gene discovery.