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