June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Method development for analysis of tear proteins using selected reaction monitoring
Author Affiliations & Notes
  • Simin Masoudi
    Brien Holden Vision Institute, Sydney, NSW, Australia
    School of Optometry and Vision Science, University of New South Wales, Sydney, NSW, Australia
  • Ling Zhong
    Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia
  • Mark Raftery
    Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia
  • Mark Willcox
    School of Optometry and Vision Science, University of New South Wales, Sydney, NSW, Australia
  • Footnotes
    Commercial Relationships Simin Masoudi, None; Ling Zhong, None; Mark Raftery, None; Mark Willcox, Allergan Inc (C), Allergan Inc (R), Brien Holden Vision Institute (P), Bausch + Lomb (C), Basuch + Lomb (R)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 930. doi:https://doi.org/
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    • Get Citation

      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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 486 cornea: tears/tear film/dry eye • 663 proteomics • 477 contact lens  
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