Abstract:

Background : The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interestingly candidate for in-depth proteomic analysis.

Results: 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 1D 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 (317 vs. 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 were previously described in the literature.

Conclusions : Equilibrium between proteases and protease inhibitors, and between oxidative reactions, is an important feature of the ocular environment. Identification of a much larger set of proteins participating in these reactions may allow discovery of molecular markers of disease conditions of the eye.