April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Protein Profiling of Meibum in Postmenopausal Normal and Dry Eye Patients
Author Affiliations & Notes
  • M. Thangavelu
    College of Optometry, College of Optometry,
    The Ohio State University, Columbus, Ohio
  • J. J. Nichols
    College of Optometry, College of Optometry,
    Ohio State University, Columbus, Ohio
  • K. B. Green-Church
    Mass Spectrometry and Proteomics Facility, Mass Spec & Proteomics Facility,
    Ohio State University, Columbus, Ohio
  • L. Zhang
    Mass Spectrometry and Proteomics Facility, Mass Spec & Proteomics Facility,
    The Ohio State University, Columbus, Ohio
  • K. K. Nichols
    College of Optometry, College of Optometry,
    Ohio State University, Columbus, Ohio
  • Footnotes
    Commercial Relationships  M. Thangavelu, None; J.J. Nichols, None; K.B. Green-Church, None; L. Zhang, None; K.K. Nichols, Consultant to Inspire, Allergan, Pfizer, Alcon, C.
  • Footnotes
    Support  NIH NEI R01 EY015519
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2373. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      M. Thangavelu, J. J. Nichols, K. B. Green-Church, L. Zhang, K. K. Nichols; Protein Profiling of Meibum in Postmenopausal Normal and Dry Eye Patients. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2373.

      Download citation file:


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

      ×
  • Supplements
Abstract
 
Purpose:
 

To analyze the protein component of the meibomian gland secretion, and to optimize mass spectrometry (MS) techniques to assess protein expression between postmenopausal (PM) normal and dry eye (DE) subjects.

 
Methods:
 

Meibum was collected using microcapillary tubes from patients categorized into normal and DE using the modified Delphi/DEWS dry eye severity grading scheme. Protein extraction optimization was performed on individual and pooled normal samples using three extraction solvents (chloroform-methanol-water; phenol-sucrose and TFA-acetonitrile) and two assays (BCA assay and CBQCA assay). PM normal and PM DE samples from two separate visits were used for MS analyses. Pools of 5 PM normal samples and 5 PM DE samples were dissolved in 6MUrea-100mM Tris buffer, followed by Trypsin (porcine) digestion and resulting peptides were analyzed by LC-MS/MS. The in-solution digestion procedure was repeated in RapiGestTMSF buffer and analyzed by LTQ Orbitrap XL (Thermo Scientific). Data search was performed against SWISS-PROT human database using MASCOT search engine.

 
Results:
 

The amount of detectable protein in individual samples was at the lower range of sensitivity of BCA assay. Average protein detected by CBQCA assay was 0.24±0.16µg per individual sample. MS analyses identified 36 proteins in normal and 27 proteins in DE with Urea buffer and 61 proteins in normal and 68 proteins in DE with RapiGestTMSF buffer. The most predominant proteins were keratins, with more keratin classes expressed in DE samples. With RapiGestTM, 30 proteins were found only in DE group and 23 were found only in normal group (Table 1).

 
Conclusions:
 

CBQCA assay is a more sensitive method for protein determination of meibum samples and confirms nanogram amounts of protein in a single capillary-collected sample. The in-solution digestion procedure has been optimized with RapiGestTMSF buffer to detect increased number of proteins from pooled meibum samples. Quantitative protein profiling by ITRAQ technique is planned.  

 
Keywords: cornea: tears/tear film/dry eye • proteomics 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×