Purchase this article with an account.
L Zhou, RW Beuerman, A Barathi, D Tan; Analysis of Rabbit Tear Proteins by High Pressure Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3140.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Tears, a complex protein mixture, are critical to good vision. In this study, we have evaluated the applicability of LC-ESI-MS to display the protein profile from rabbit tears. The response of the tear protein profile to corneal wound healing in a rabbit model was investigated. Methods: Tears were collected from New Zealand White rabbits prior to and daily for four days following a unilateral 5mm dia. epithelial wound using 10 µl calibrated glass microcapillary tubes. Tear proteins were eluted by a reverse-phase HPLC column and the tear protein profile was monitored by ESI positive TIC (Total Ion Current) chromatography. Results: Tear proteins could be separated into 18 peaks, each of which contained a number of protein components. The molecular size of each protein component was determined by on line electrospray ionization (ESI) mass spectrometer. Major tear protein components, lactoferrin, lysozyme (minimally detectable in rabbit tears), albumin, lipocalin, lipophilin and ß2-microglobulin were rapidly identified by this method. Based on the mass data, ß2-microglobulin revealed glycosylation with N-acetyl-hexsamine. ESI positive TIC chromatograms and mass spectra indicated differences in tear protein profile before and after corneal wounding. After the cornea was wounded for 24 hours, the level of a protein with molecular weight of 14717 Da was found to be 7-fold higher than that in control tears. It dropped back to normal levels 96-hours after wounding. Conclusion: The LC-ESI-MS has been demonstrated to be a reproducible, fast and simple method for identification and analysis of many protein components of tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping peptides and small proteins (< 80 kDa) in tears and should be useful to examine tear protein levels in experimental condition.
This PDF is available to Subscribers Only