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C. K. Bahler, M. C. Charlesworth, B. J. Madden, K. G. Howell, K. A. Cook, M. P. Fautsch; Proteome Analysis of Human Aqueous Humor. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1642. doi: https://doi.org/.
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The trabecular meshwork receives nutrients and external signals from aqueous humor, a complex mixture of electrolytes, organic solutes, growth factors, and additional proteins. Characterization of the protein component in aqueous humor will help to identify molecules involved in maintaining a homeostatic environment to anterior segment tissues such as the trabecular meshwork. To understand the protein complexity, we analyzed the proteome of normal human aqueous humor.
85 aqueous humor samples obtained during cataract surgery were divided into 3 groups (mean age 72 years; mean protein concentration 220 ng/µl) and immunodepleted of 6 abundant proteins (albumin, IgG, IgA, haploglobin, antitrypsin, transferrin). Remaining proteins were separated by SDS-PAGE and silver-stained. 33 gel slices were excised from each group (n=99), digested with trypsin, and subjected to LC/MS/MS. Mascot search algorithm matched MS/MS spectra to peptides in Swissprot human database. Proteins with ≥ 2 peptide matches (MS/MS ion score >30) and identified in 2/3 samples were defined as positive. An additional 21 aqueous humor samples were divided into 4 groups and used to probe protein arrays (RayBiotech, Inc.). Proteins with a signal >3 fold above negative control in at least 3/4 groups were defined as positive.
A total of 467 proteins were identified in human aqueous humor. Only 15 proteins were found by both techniques. Of the 270 proteins identified by LC/MS/MS, only 10% have been identified in aqueous humor, and only 57% have been reported in human plasma. The majority of these proteins had catalytic, enzymatic or structural functions. Of the 197 growth factors, cytokines, and receptors identified by protein arrays, >80% are newly identified in human aqueous humor. These include members of the TGF-β and TNF superfamilies.
Many of the proteins identified in human aqueous humor are not in plasma or are in different proportions; emphasizing the fact that aqueous humor is a unique nutrient source for anterior segment tissues. The elucidation of the normal aqueous humor proteome will enable future studies to identify proteins that are differentially expressed in glaucomatous aqueous humor.
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