Proteomic analysis of aqueous humour from patients with acute corneal rejection

Abstract

ABSTRACT.Purpose: To compare the basic proteomic composition of aqueous humour (AH) from patients with corneal rejection (patients) with AH from patients with cataract (controls). Methods: Aqueous humour was analysed for total protein concentration using Bradford's method and for protein composition using two-dimensional (2D) gel electrophoresis. Image analysis was used to detect protein spots in 2D gels that were increased by more than factor 2 in patients as compared with controls. Increased spots were identified by immunoblotting and mass spectrometry. Results: Aqueous humour from patients contained significantly higher total protein concentration than did AH from controls. A total of 31 spots were significantly increased in 2D gels from patients. The spots were derived from albumin, a1-antitrypsin, apolipoprotein J, cytokeratin type II, serin proteinase inhibitor and transthyretin. After correction of spot volumes by total protein concentrations, 10 spots derived from albumin, cytokeratin type II and a1-antitrypsin remained significantly increased. Conclusion: The proteomic composition of AH differed significantly between patients and controls. The identified proteins suggest that the changes in AH are due to at least three different mechanisms: breakdown of the aqueous-blood barrier, enzymatic degradation, and liberation of locally synthesized proteins.

Proteomics in ocular fluids

“…Funding et al analyzed the proteins in aqueous humor samples of patients with corneal rejection with 2-DE. They found a significant difference in the protein composition in patients in comparison to control subjects, some of the increased protein spots derived from albumin, cytokeratin type II, and a1-antitrypsin [43]. The authors suggest a breakdown of the aqueous-blood barrier, an enzymatic degradation, or perhaps liberation of locally synthesized proteins as a possible explanation for this phenomenon.…”

“…Since the total volume of aqueous humor in the anterior chamber is around 150-200 mL [41], and even decreases with age [42], it is hardly possible to obtain more than 100-150 mL of the sample from one subject [43,44].…”

“…Additionally, MS techniques can be used for the proteomic analysis of ocular fluids like ESI-MS or MALDI-MS. A characterization of protein patterns of aqueous humor of patients with corneal rejection and cataract led to the identification of 31 significantly increased proteins in corneal rejection patients [43]. These identifications were based on peptide mass fingerprints with MALDI-TOF-MS.…”

Multidimensional Protein Identification Technology

“…In 2005, Funding et al [15] used two-dimensional electrophoresis (2-DE) to separate AH proteins and image analysis to determine differentially expressed AH proteins in patients with acute corneal rejection. Then using a combination of immunoblotting, matrix-assisted laser [21].…”

“…In 1998 Rohde et al [5] used a proteomic approach to analyze AH proteins but because of technological limitations, proteins were identified based solely on molecular weight and thus must be considered tentative. 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.…”