Proteomic analysis of the tear film in patients with keratoconus

PURPOSE To identify proteins differentially expressed between the tear film of keratoconus (KC) patients and control subjects using two dimensional electrophoresis (2-DE) and mass spectrometry-based techniques. METHODS Twenty two patients (44 eyes) diagnosed with bilateral KC and 22 control subjects (44 eyes) were studied in a prospective case-control study. Keratoconus screening programs and Orbscan II topographies were performed on all participants. Tear samples were collected by the Schirmer I method using filter paper. Proteins were extracted from the Schirmer strips and separated by 2-DE. Comparison of protein patterns was performed using PDQuest Software and protein differences were identified by mass spectrometry. Finally, results were validated by western-blot. RESULTS Four spots were identified to be differentially expressed between KC patients and control subjects. Three of them were more expressed in healthy subjects and they were identified as zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain). The other spot was more expressed in KC patients and it was identified as ZAG. Differences in ZAG seem controversial in two different spots because different posttranslational modifications, however, analysis of both spots revealed that globally, ZAG is overexpressed in healthy subjects. Founded differences in ZAG, lactoferrin, and IGKC expression were subsequently validated by western blot. CONCLUSIONS IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects. These differences could contribute to the knowledge of the pathophysiology of this disease.