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Ocular Surface  Dry Eye Disease Update on Human Tear Proteome Piera Versura and Emilio C Campos 1. Lab Head, Laboratory for Ocular Surface Analysis; 2. Emilio Campos, Professor and Chief of Ophthalmology, Ophthalmology Unit, Alma Mater Studiorum University of Bologna, S Orsola-Malpighi Teaching Hospital, Bologna, Italy Abstract Tear film is a thin layer of non-newtonian fluid covering and protecting cornea and conjunctiva epithelia. The multifaced functions of tears are reflected by their complex structure and advances in proteomics/metabolomics/lipidomics technologies have greatly deepened the knowledge of the chemical composition of tears. The purpose of this article is to survey recent advances in proteomic analysis of human tears and to summarise the most relevant proteins proposed as biomarkers in dry eye. Besides, the potential clinical application of these biomarkers is discussed. Keywords Human tears, dry eye, proteomic analyisis Disclosure: The authors have no conflicts of interest to declare. Received: 10 January 2013 Accepted: 5 February 2013 Citation: European Ophthalmic Review, 2013;7(1):36–41 Correspondence: Piera Versura, Ophthalmology Unit, University of Bologna, Policlinico S Orsola-Malpighi, Pad 1 Palagi, Via Palagi, 9 40138 Bologna, Italy. E: piera.versura@unibo.it Tears are a critical body extracellular fluid coating surface epithelial cells of cornea and conjunctiva and providing the optically smooth surface necessary for refraction of light onto the retina. Production and quality of tears are controlled and co-ordinated by the lachrymal function unit (LFU), which consists of the main and accessory lachrymal glands, the ocular surface (including the cornea and conjunctiva), the meibomian glands and the interconnecting neural pathways. 1 A great body of past literature established that the conditions of LFU are reflected in the composition of tears. Proteomics is the research area studying proteins expressed in a given biological compartment at a given time. The original definition of proteins as products of gene expression has lately included post-translational modifications of these gene products occurring in cell metabolism and turnover. 2 These arguments and the concept of temporal dynamics are of specific relevance in a body fluid as tears, directly in tiny equilibrium with the external environment and continuously exposed to both internal and external noxious agents. Proteomic studies rely upon high-tech separation methods and equipment resolving the biological sample complexity, with the aid of procedures able to remove abundant proteins from samples (such as albumin and immunoglobulins in sera) and exhibit less abundant proteins. It is likely, however, that this pre-analytical step removes other possibly important protein biomarkers. An alternative option is to analyse body fluids closer to the site of interest: tears are a useful and accessible source for evaluating ocular surface tissue and lachrymal gland function in a number of disease conditions, such as dry eye, as well as treatment response. Tears contain proteins, peptides, lipids, small molecule metabolites, electrolytes and an increasing attention in human tear proteome has been devoted in recent years, with the aim to develop biomarkers of disease. 3–5 36 In this article we summarise the most relevant literature in proteomic analysis of human tears and point out those proteins proposed as biomarkers in dry eye. Potential application of tear proteomics in clinical setting and the related difficulties are also discussed. Non-invasive Tear Sampling and Storage Standardisation of tear collection was and still is a major hindrance for investigators. The main pitfalls are related to sampling of the needed amount of tears possibly avoiding unwanted dilution as a consequence of reflex production. It is recognised, in fact, that stimulated and unstimulated tears show different protein composition as demonstrated for secretory IgA and albumin. 6 A comparison of different tear collection methods is provided in Zhou, 2012. 7 Methods currently available are based upon collection from Schirmer strips or various types of collectors (like sponges or rods positioned in the conjunctival meniscus to be impregnated by tears). Both are widely used methods but still unstandardised, since different cellulosic or polymeric materials are used, each having different adsorption, adhesive and releasing characteristics. In addition, many cell proteins can adhere (in particular, keratins) which may mask other protein peaks and quantification of proteins expressed /ml sample can turn to be difficult. Perhaps collection by direct aspiration of either basal or flushed tears would be the most appropriate method. It can be accomplished through glass capillaries or a micropipette with a disposable sterile minitip at the outer conjunctival canthus. 8 The method is considered by many to be time-consuming, impractical and uncomfortable but after a short amount of training it can be safely performed by any operator. Tears contain various proteolytic enzymes, therefore appropriate storage is important to avoid sample modifications and unreliable results. It has been suggested that tears can be maintained without significant changes up to one week at 4 °C, up to two months at -20 °C, and up to four months at -70 °C before analysis. 9 © Tou c h ME d ic al ME d ia 2013