Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

In this study, we employ state-of-the-art mass spectrometric identification, using both a hybrid linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer, and high confidence identification by two consecutive stages of peptide fragmentation (MS/MS/MS or MS3), to characterize the protein content of the tear fluid. Low microliter amounts of tear fluid samples were either pre-fractionated with one-dimensional SDS-PAGE and digested in situ with trypsin, or digested in solution. Five times more proteins were detected after gel electrophoresis compared to in solution digestion (320 versus 63 proteins). Ontology classification revealed that 64 of the identified proteins are proteases or protease inhibitors. Of these, only 24 have previously been described as components of the tear fluid. We also identified 18 anti-oxidant enzymes, which protect the eye from harmful consequences of its exposure to oxygen. Only two proteins with this activity have been previously described in the literature.Interplay between proteases and protease inhibitors, and between oxidative reactions, is an important feature of the ocular environment. Identification of a large set of proteins participating in these reactions may allow discovery of molecular markers of disease conditions of the eye.The eye is covered by a thin, fluid film that serves several functions. It has critical roles in the optical system, lubricates the eye, provides nutrients and growth factors to the epithelium and serves as a barrier to the outside environment [1,2]. In the last function, it protects the eye against infection. The tear film is an aqueous layer containing proteins and electrolytes secreted by the lacrimal gland situated within the orbit above the lateral end of the eye, and other accessory gland secretions. Additionally, tear fluid is in contact with the epithelium of the lid and, thereby, is in indirect contact with the blood circulation. Major tear proteins