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J.E. Evans, K.M. Green, R.M. Sullivan, D.A. Schaumberg, M.R. Dana, D.A. Sullivan; Proteomic Analysis of Human Meibomian Gland Secretions . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2478.
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Purpose: Researchers have recently identified over 490 proteins in the human tear film. We hypothesize that many of these proteins originate from the meibomian gland, and influence both tear film stability and ocular surface integrity. Our rationale is that the meibomian gland is a large sebaceous gland, and sebaceous glands are known to secrete a variety of proteins (e.g. IgA, pro-inflammatory cytokines) and lipids in non-ocular sites. The purpose of this study was to develop the proteomic methodology to permit the testing of our hypothesis. Methods: Meibomian gland secretions were collected from the left and right lower eyelids of male and female volunteers (n = 7) and placed in a chloroform-methanol mixture. Samples were processed by partitioning in a chloroform-methanol-water biphasic system. The upper non-lipid phase materials were reduced with dithiothreitol, alkylated using iodoacetamide, and trypsin digested in 8 M urea to obtain peptides for microcapillary (µ) HPLC/mass spectrometry (MS)/MS protein identification. This peptide mixture was separated by µ-capillary reverse phase chromatography and the effluent analyzed by online MS and MS/MS using an ion trap mass spectrometer. The MS/MS spectra from the tryptic peptides detected were used to identify the proteins present in the samples using SEQUEST software for comparison to protein databases. Results: Our methodologic approach to date has permitted the identification of over 15 proteins in meibomian gland secretions. Proteins include Tro alpha 1 H (an IgA heavy chain variant), farnesoid-X receptor (a regulator of cholesterol homeostasis), megakaryocyte associated tyrosine kinase and keratin 10. However, we have yet to completely optimize our analytical procedures (e.g. for removing salts, reducing possible interference by glycosylated peptides), or to apply more sensitive and specific µLC/µLC/MS/MS techniques (MudPIT). Conclusions: . Our findings indicate that the meibomian gland may secrete a number of proteins into the tear film. (Supported by research grants from Allergan, NIHEY05612 and the SERI/MEEI Joint Clinical Research Center)
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