A technique for shotgun proteomic analysis of the precorneal tear film in dogs with naturally occurring primary glaucoma

Objective To introduce a protocol for the characterization of protein patterns in tears of dogs with primary angle closure glaucoma (PACG) and primary open-angle glaucoma (POAG). Animals Nineteen dogs (25 eyes). Methods Tear samples were collected using a Schirmer tear strip, from dogs with PACG (PACG-affected eyes, n = 8; unaffected eyes predisposed to PACG, n = 7), POAG (n = 4), and healthy controls (n = 6). Protein precipitation and trypsin digestion were performed for analyses via liquid chromatography-tandem mass spectrometry. Proteins were identified using the SwissProt protein sequence database. Relative protein expression in 17 eyes (15 dogs) was evaluated using Proteome Discoverer 2.0. Pathway analyses were performed to investigate molecular mechanisms associated with primary glaucoma. Results Unique peptides were identified in 505 proteins, with Major allergen Can f 1 and albumin identified with high confidence. Proteins unique to tears from diseased eyes (PACG: n = 7; POAG: n = 14) were identified. Nucleoside diphosphate was unique to tears in PACG eyes naive to therapy, while retinal binding protein and NSFL1 cofactor p47 were unique to medicated PACG eyes. Relative expression of 34 proteins differed between disease states. Pathway analyses identified that the 'inflammatory response' was among the top disease/disorders in dogs with primary glaucoma (PACG and POAG) but not in healthy controls. Conclusion Tear samples suitable for mass spectrometry were readily obtained from pet dogs without needing specialized equipment. Further studies to validate the findings and explore potential candidate biomarkers for early disease detection and potential therapeutic targets are indicated.