The aim of this study was to develop a fast and reliable analytical procedure for the display of the
protein components of tears that can be used to differentiate the status of the ocular surface. Using this new procedure, we analyzed the tear
protein components following a corneal
wound in the rabbit. Calibrated 10-microL glass, fire-polished capillary micropipettes were used to collect tears from New Zealand White rabbits prior to and daily for 9 days following a unilateral 6-mm diameter centrally placed anterior
keratectomy.
Tear proteins were eluted by a reversed-phase high-performance liquid chromatography (RP-HPLC) column and the tear
protein profile was monitored by electrospray ionization (ESI) mass spectrometry positive total ion current (
TIC) chromatography.
Tear proteins were reliably separated into 17 peaks, each of which contained one or a number of
protein components. The molecular weight of each
protein component was determined by on-line ESI. Major tear
protein components,
lactoferrin,
lysozyme (minimally detectable in rabbit tears),
albumin,
lipocalin, lipophilin and beta2-microglobulin, were tentatively identified by this method. Based on the mass spectrometric data, beta2-microglobulin was found to be glycosylated with N-acetylhexosamine. ESI-positive
TIC chromatograms and mass spectra revealed comparative differences in the tear
protein spectra after corneal wounding. One day after wounding, rabbit
lysozyme with a molecular weight of 14,717 Da was found to be 8-fold higher in the tears of wounded eyes when compared with tears from unwounded eyes. It dropped back to normal 3 days after wounding. The expression of an unidentified tear
protein with the molecular weight of 16,060 Da was also elevated after corneal wounding and returned to normal level by day 5. In this study, LC/ESI-MS was developed as a fast, reproducible and simple method for the identification and analysis of many of the
protein components of the tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping
peptides and
proteins (<80 kDa) in tears.