Characterization of human neutrophil peptides (α-Defensins) in the tears of dry eye patients

The potential peptide biomarkers of dry eye disease were rapidly and sensitively screened in tears by simple sample pretreatment and modern mass spectrometry. The tear sample was collected by simply wiping the corner of the eye with a small piece of Schirmer filter paper from the patients confirmed to have dry eye disease and from normal controls. The peptides adsorbed on the filter paper were extracted by vortexing it with sinapinic acid solution in a vial. A drop of the extracted solution was then deposited on the sample plate and examined using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) without any other sample pretreatment. We found human neutrophil peptides (HNPs), so called alpha-defensins 1-3, which are potential biomarkers for inflammation, were significantly up-regulated in the tears collected from all the dry eye disease patients. The identity of the alpha-defensins in human tears was confirmed through (i) matching their molecular ions with those of standards, (ii) literature searches and (iii) electrophoretic purification and separation of the tears followed by in-gel tryptic digestion and MALDI-TOF analysis. The presence of large quantities of HNPs in the tears of dry eye patients suggests the occurrence of chronic inflammation in the ocular regions of dry eye patients.