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M. Bortolotti, A. Palmigiano, A. Messina, E. Mazzola, S. Milazzo, D. Garozzo, A. Leonardi; Identification of Tear Fluid Biomarkers in Vernal Keratoconjunctivitis Using iTRAQ Quantitative Proteomics. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1931.
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The aim of the study was to analyze and compare the quantitative proteomic profile of human tears in vernal keratoconjunctivitis (VKC) patients and healthy subjects using LC MALDI mass spectrometry and iTRAQtm in order to identify biomarkers for the disease.
Using a glass microcapillary tube, tears were collected from healthy subjects (n=9) and VKC patients (n=11). Digested samples were treated with iTRAQtm (Isobaric Tag for Relative and Absolute Quantitation). Separation of tryptic peptides was realized using a µHPLC interfaced with a fraction collector (DIONEX). MS and MS/MS mass spectra were performed using a MALDI TOF/TOF 4800 Applied Biosystem spectrometer using as a matrix solution of CHCA (α-Cyano-4-hydroxycinnamic acid) 3mg/ml in ACN:H20 3:1 containing 0.15 mg/ml of diammonium citrate. Protein PilotTM software with ParagonTM algorithm v3.0 or GPSTM with Mascot Engine (Matrix Science) were used as search engines with SwissProt or IPI human as the databases.
VKC spectra showed similar profiles. A significant number of peptides were examined and 42 proteins were successfully identified. In all VKC samples, high levels of serum albumin were found, up to 60 times higher than control levels. Tear levels of serum albumin were correlated to the severity of disease. A different up- or down-regulation of several proteins was identified in healthy and pathological samples. In VKC, increased levels of lysozyme C, extracellular glycoprotein lacritin and proline-rich 4 protein levels were found, as well as an imbalance between proteases and protease inhibitors.
This technique was useful for the identification of biomarkers in human tears. Analysis allowed for identification of previously undetected factors in tears of VKC patients. Quantitative differences between normal and VKC samples may find application in the diagnosis of different forms of ocular surface inflammation.
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