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Tatiana Maria Suarez-Cortes, Javier Soria, Arantxa Acera, Nerea Gonzalez, Ibon Iloro, Felix Elortza, Jesus Merayo-Lloves, ; Human tear peptide/protein profiling study of Keratoconus grades by SPE-MALDI-TOF Mass Spectrometry analyses. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2006.
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Changes in tears of Keratoconus (KC) patients have been widely reported, however, peptide/protein profiling has not been widely approached. The goal of this study was to analyze the tear peptidome/protein profiles (below 20kDa) of KC patients and to determinate their relation with clinical grades of the disease. Secondly, this work aimed to evaluate whether the tear profiling allows a categorization of patients which would be potentially useful for early diagnosis and disease grading.
A total of 72 KC patients (20 mild, 21 moderate and 31 severe) and 14 healthy subjects were studied. The biomicroscopy exam was performed to detect signs of KC and corneal topography study was used to quantify topographic parameters. The modified Rabinowitz McDonnell test was used to confirm KC diagnosis and KC severity was classified according to the steepest simulated keratometry reading on the keratometric map (K2 <45 D, mild; K2 between 45 -52 D, moderate; K2 >52 D, severe KC). Tear samples were collected by capillary and solid-phase extraction using magnetic beads (MB) with reverse phase affinity (C18), in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) was used for human tear peptide/protein profiling analysis. Spectra obtained in the range of 1000-20000 Da were pre-processed and analyzed using multivariate statistical techniques.
The tear peptide/protein profiling revealed differentially expressed peaks depending on grade severity of KC. From 243 peaks detected, 43 presented intensities significantly changed. Canonical discriminate analysis using these 43 most discriminate variables showed a good spatial separation of groups based on tear profile indicating a high correlation between peptide/profile and disease severity.
Tear peptide/proteome profiling of KC patients analyzed by SPE-MALDI-TOF allows classifying of patients according to clinical severity with high accuracy. Early diagnosis of KC patients is possible based on tear peptidome/protein profile changes. MALDI-TOF profiling of tears constitutes a sensitive method for grading severity of KC and for identifying outliers. Finally, peptidome/protein patterns may provide opportunities for research into the pathogenesis of the disease and to improve diagnosis, prognosis and monitoring of response to therapies in clinical trials.
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