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Simin Masoudi, Ling Zhong, Mark Raftery, Mark Willcox; Method development for analysis of tear proteins using selected reaction monitoring. Invest. Ophthalmol. Vis. Sci. 2013;54(15):930. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies of dry eye and contact lens (CL) related dry eye have shown qualitative differences in lactoferrin, lysozyme, lipocalin 1, prolactin-induced protein and proline rich protein 4. The aim of this study was to develop a method for simultaneous detection and accurate quantification of these five proteins in human tears using selected reaction monitoring (SRM) mass spectrometry.
Basal tears were collected from normal subjects (male=1, female=6, CL=3, No CL=4).Tears were processed as described before with modifications. SRM transitions from two peptides representing each protein were selected using Skyline software then analyzed using nano-flow liquid chromatography mass spectrometry. Calibration standards were generated using unlabelled synthetized peptides diluted to a range of 1 to 1000fmol/µL in the presence of a fixed amount of stable-isotopically labelled peptides (50fmol/µL). The ratios of the peak areas of the unlabelled/labelled peptides were plotted against the concentrations of the corresponding unlabelled peptides. The concentration of endogenous proteins (µg/µL) in tear samples was deduced using the peak area ratio of the endogenous peptides to labelled peptides.
The limits of quantification for the selected peptides were below 50pg/μl. The recovery of peptides from spiked digested tears was ≥56% and the coefficient of variation values were ≤16% which show good precision of the method. For lactoferrin (1.20±0.77μg/μL), lysozyme (2.11±1.50μg/μL) and lipocalin-1 (1.751±0.99μg/μL) the findings were consistent with findings from previous ELISA studies. Tear levels of prolactin-induced protein (0.09± 0.06μg/μL) and proline rich 4 (0.80±0.50μg/μL) are reported here for the first time.
SRM method can be used for simultaneous detection and quantification of the five selected proteins in low volume human tear samples (2.5µl per sample) without prior purification of each protein component or need for antibodies.
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