June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Multiplexed Quantitative Analysis of 51 Proteins in Human Tears using High Resolution Multiple Reaction Monitoring (HR-MRM) Mass Spectrometry
Author Affiliations & Notes
  • Lei Zhou
    Singapore Eye Research Institute, Singapore, Singapore
    Ophthalmology, National University of Singapore, Singapore, Singapore
  • Louis Tong
    Singapore Eye Research Institute, Singapore, Singapore
  • Roger Beuerman
    Singapore Eye Research Institute, Singapore, Singapore
    Ophthalmology, National University of Singapore, Singapore, Singapore
  • Footnotes
    Commercial Relationships Lei Zhou, Singapore Eye Research Institute (P); Louis Tong, None; Roger Beuerman, Allergan (F), SERI (P), Santen (R)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2195. doi:
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    • Get Citation

      Lei Zhou, Louis Tong, Roger Beuerman; Multiplexed Quantitative Analysis of 51 Proteins in Human Tears using High Resolution Multiple Reaction Monitoring (HR-MRM) Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2195.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Tear proteins are intimately related to the pathophysiology of the ocular surface of the eye. Many studies have demonstrated that the tear fluid is an accessible and useful source in studying ocular surface disorders and biomarker discovery. This study describes the use of a high resolution MRM approach to develop assays for biologically important tear proteins.

Methods: Human tear samples were collected from 1000 consenting patients with no eye complaints (411 male, 589 female, average age 55.5 years, SD 14.5 years) using the Schirmer tear test strips and pooled into a single global control sample. Quantification of proteins is carried out by selecting “signature” peptides derived by trypsin digestion of the target protein. A 2-hour nanoLC-MS/MS run was used to separate the tryptic peptides and perform quantitation of human tear proteins in HR-MRM mode. Samples were analyzed in triplicate. Twenty-one high abundant proteins were further accessed for signal reproducibility.

Results: Fifty-three peptide assays that represent 51 high and intermediate abundant tear proteins were developed. All assays showed consistent retention time with a coefficient of variation (CV) of less than 2%. As for peak area, 17 out of the 21 assays showed a reproducible peak area with CV less than 20%. Some well characterized tear proteins, lacritin, mammaglobin-B, S100A4, S100A8, and prolactin inducible protein (PIP) showed the highest reproducibility of peak area, with CV less than 2%.

Conclusions: These multiplexed MRM-based assays show great promise to be further developed for biomarker validation in human tear samples.

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