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K. T. Ude, N. Boehm, M. Wiegand, N. Pfeiffer, F. H. Grus; Dig Deeper Into the Tear Proteome: Advanced Protein Profiling in Dry Eye Patients. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4140.
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The protein and peptide composition of tears plays an important role in inflammatory processes of the ocular surface. In previous studies it was shown that a change in expression levels of several tear proteins in patients suffering from dry eye diseases is detectable. Aim of this study was to perform a proteome wide, mass spectrometry based analysis of tear proteins, and to compare patterns of patients with different dry eye subtypes to those from healthy subjects.
Healthy subjects (CO, n=6), aqueous deficient dry eye patients (DryAq, n=3) and patients with an aqueous deficiency and changes in the lipid phase (DryAqLip, n=3) were analyzed. Tears were collected using Schirmer strips. Proteins were eluted and separated by 1D SDS-PAGE. The lanes were cut into 32 parts followed by in gel digestion. Peptide solutions were fractionated on a robot station using C18 chromatography, followed by mass spectrometry analysis (MALDI-TOFTOF MS). Biomarker detection was performed using various algorithms like ANOVA, multivariate statistics, and artificial neural networks.
A total of 331 different proteins could be identified, including high abundant tear proteins but also proteins related to inflammatory processes (e.g. TNFα) or signaling pathways (GTP-binding proteins). The statistical analysis revealed several significant differences between tear protein profiles of healthy subjects and dry eye patients (P≤0.01), and moreover between the dry eye subtypes (P≤0.01). E.g. we found a 6 fold increase of secretoglobin in DryAq subjects (CO: ME=30, DryAq: ME=191). Using artificial neural networks we were able to classify patients by means of their protein profiles with a specificity and sensitivity of ≥90% (AUC of ROC-curve r≥0.9).
Using a sophisticated mass spectrometry approach we could improve the tear protein profiling leading to a high number of identified proteins. Besides validation of biomarkers known from previous studies we detected several new proteins with increased or decreased amounts in the tears of dry eye patients. Especially proteins functionally related to inflammatory processes as well as signaling proteins were affected by these alterations. The detected markers may contribute to the development of innovative drug targets as well as improved diagnostic tools.
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