Purpose: Protein and peptides in tears play an important role in ocular surface diseases. In previous studies, changes have been demonstrated in the electrophoretic protein profiles of patients with dry eye. The purpose of this work was to determine the usefulness of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip Array (Ciphergen Biosystems, Inc., Fremont, CA) technology for the automated analysis of proteins and peptides in tear fluid.
Methods: Patients with dry eye (DRY, n = 88) and healthy subjects (CTRL, n = 71) were examined. Their tear proteins were analyzed using SELDI-TOF-MS ProteinChip Arrays with three different chromatographic surfaces (CM10 cation exchange, Q10 anion exchange, and H50 reversed-phase) prepared by means of a laboratory liquid-handling robotic workstation. The data were analyzed by multivariate statistical techniques and artificial neural networks, and the most important biomarkers were purified and identified by tandem MS.
Results: Complex patterns of tear proteins and peptides were detected. The different chromatographic surfaces revealed the selective enrichment of proteins such as lipocalin and lysozyme. Discriminant analysis demonstrated highly significant changes in the protein profiles in patients with dry eye (P < 0.001). With a seven-peptide multimarker panel, an artificial neural network could differentiate between patients with dry eye and healthy individuals with a specificity and sensitivity of 90%. The identification of biomarkers revealed an increase of inflammatory markers in patients with dry eye and a decrease of some proteins that may have protective functions.
Conclusions: The SELDI-TOF-MS technology seems to be ideally suitable for the mass screening of peptides and proteins in tears. This highly sensitive approach dramatically reduces the analysis time and provides protein profiles with great mass accuracy. Thus, it may become a very useful tool in the search for potential biomarkers for diagnosis and new therapeutics in ocular diseases such as dry eye.