Identification of Human Tear Fluid Biomarkers in Vernal Keratoconjunctivitis Syndrome Using iTRAQ Quantitative Proteomics

Introduction Vernal keratoconjunctivitis (VKC) is a recurrent or chronic ocular allergic disease that affects mostly children and young adults living in warm climates worldwide. Understanding and treating VKC has been a challenge for ophthalmologists since the pathogenesis is unclear and anti-allergic therapy often unsuccessful. Quantitative and qualitative characterization of tears proteoma in VKC patients may be useful for identifying disease markers and may find application in the diagnosis and management of this disease. The aim of this study was to analyze peptide profiles in human tears by using LC MALDI mass spectrometry and iTRAQ to elucidate compositional differences between healthy subjects and patients affected by VKC. Methods Using a glass microcapillary tube, tears were collected from healthy subjects (n=9) and VKC (n=11). Tear samples were digested with trypsin and treated with iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) using manufacturer's protocol. The separation of the peptidic fragments were realized by a µHPLC DIONEX (LC Packings) system. The MS and MS/MS mass spectra were performed using a MALDI TOF/TOF 4800 Applied Biosystem spectrometer. Data analysis was performed using Protein Pilot(TM) software with Paragon(TM) algorithm v3.0 search engine (Applied Biosystems) and GPS(TM) software (Applied Biosystems) with Mascot search engine and searched against the SwissProt and the IPI (International Protein Index) databases. Preliminary data This technique was found to be very useful in the characterization of the proteoma of human tears. Differences were found between normal and VKC samples. In all VKC samples, high levels of albumin and other serum proteins were found in pathological subjects, up to 60 times higher than the control samples levels, indicating leakage of serum proteins into tears. We found that quantification of albumin can be used as a measure of the severity grade of this disease. For other proteins there is a percentage of up- or down-regulation between healthy and pathological samples. It seems to be also a correlation between lysozime C, extracellular glycoprotein lacritin and proline-rich 4 protein levels, and an imbalance in proteases and proteases inhibitor level. Some of these proteins are expressed differently in other pathologic eye conditions, for example, proline-rich protein 4 is down-regulated in dry eye. Identifying disease markers may find application in the diagnosis of different ocular surface inflammations. Novel Aspect High sensitive method, small amounts of sample required, no pool of different subjects needed.