advertisement
Abstract #18233 Published in IGR 9-2
Proteomic analysis of exfoliation deposits
Ovodenko B; Rostagno A; Neubert TA; Shetty V; Thomas S; Yang A; Liebmann J; Ghiso J; Ritch RInvestigative Ophthalmology and Visual Science 2007; 48: 1447-1457
PURPOSE: To increase knowledge of the biochemical composition of lenticular exfoliation material (XFM) by using proteomic approaches. METHODS: Anterior lens capsules from patients with and without exfoliation syndrome (XFS) were homogenized in formic acid and subjected to cyanogen bromide (CNBr) cleavage, and the pattern of chemically generated fragments was compared by SDS-PAGE after silver staining. Unique XFS bands not present in control cases were excised, digested with TPCK-trypsin, and the resultant peptides sequenced with quadrupole time-of-flight mass spectrometry (MS). In parallel experiments, CNBr-fragmented XFM was separately digested in solution with trypsin and elastase, and the resultant peptide mixture was analyzed by liquid chromatography coupled to tandem MS followed by identification through homology searches at nonredundant protein databases. Immunolocalization of the MS-identified components were performed in XFS versus control samples by using conventional immunohistochemical methods and light microscopy. RESULTS: In addition to fibrillin-1, fibronectin, vitronectin, laminin, and amyloid P-component, which are well-known extracellular matrix and basement membrane components of XFM, the proteomic approaches identified the multifunctional protein clusterin and tissue inhibitor of metalloprotease (TIMP)-3 as well as novel molecules, among them fibulin-2, desmocollin-2, the glycosaminoglycans syndecan-3, and versican, membrane metalloproteases of the ADAM family (a disintegrin and metalloprotease), and the initiation component of the classic complement activation pathway C1q. In all cases, classic immunohistochemistry confirmed their location in XFM. CONCLUSIONS: A novel solubilization strategy combined with sensitive proteomic analysis emphasizes the complexity of the XFS deposits and opens new avenues to study the molecular mechanisms involved in the pathogenesis and progression of XFS.
Dr. B. Ovodenko, Department of Ophthalmology, New York Eye and Ear Infirmary, New York, NY, USA
Classification:
3.12 Proteomics (Part of: 3 Laboratory methods)
9.4.4.1 Exfoliation syndrome (Part of: 9 Clinical forms of glaucomas > 9.4 Glaucomas associated with other ocular and systemic disorders > 9.4.4 Glaucomas associated with disorders of the lens)
