Proteomics of the aqueous humor in healthy New Zealand rabbits

Abstract

There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.

Multidimensional Protein Identification Technology

“…Thus, the next clear step in AH analysis was an extensive investigation of the human AH proteome. We could only confirm 21 of the 98 rabbit AH protein identifications reported by Stastna et al [1] in the human AH. These proteins are highlighted in Table 2 and Table 3 with the exception of the following six proteins which were identified outside of priority 1 (priority 2-4, less confident identifications): type II collagen, desmocollin, desmoglein, plasminogen, cathepsin, and retinol-binding protein.…”

“…In 2007, Stastna et al [1] employed an elegant combination of 1-DE, 2-DE, and reversed phase liquid chromatography (RPLC) for protein separation and MALDI-TOF MS and LC-MS/MS for AH protein identification. This appears to be the only publication dealing exclusively with the AH proteome, rather than a differential expression analysis, and is thus the most relevant published report of an extensive characterization of the proteome.…”

“…In 2005 Funding et al [15] and in 2008 Duan et al [16] each used twodimensional electrophoresis in combination with tandem mass spectrometry to identify seven unique AH proteins with high confidence, and in each study interfering high abundance proteins such as albumin limited the depth of the analysis. Also, in 2007 Stastna et al [1] used an elaborate combination of proteomic techniques to identify almost a hundred proteins from rabbit AH; however, the rabbit genome is incomplete so tentative cross-species identifications had to be made using a broad mammalian database. Here we employed an albumin/ IgG depletion strategy to remove these interfering abundant proteins in human derived AH, thus yielding two fractions: albumin-bound and albumin-depleted.…”

“…The AH was abundant in antioxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states.The aqueous humor (AH) is a clear fluid that fills the anterior segment of the eye and bathes the lens, iris, and corneal endothelium [1]. It is secreted by the ciliary body and functions to provide nutrients and remove waste from avascular tissues [2], as well as create the intraocular pressure that maintains the convex shape of the cornea.…”

“…The aqueous humor (AH) is a clear fluid that fills the anterior segment of the eye and bathes the lens, iris, and corneal endothelium [1]. It is secreted by the ciliary body and functions to provide nutrients and remove waste from avascular tissues [2], as well as create the intraocular pressure that maintains the convex shape of the cornea.…”