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J. Obidike, K. Schey; Characterization of the Lens Specific Cytoskeletal Protein Filensin via a Modern Proteomics Approach. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4077.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to utilize a modern proteomics approach to characterize the structure and modifications to the lens specific cytoskeletal protein filensin to begin to identify age-related alterations and as a first step in understanding its role as a binding partner to aquaporin 0.
Bovine lenses were decapsulated and dissected into nuclear and cortical regions and, after homogenization, the homogenates were separated into water soluble (WSF) and insoluble fractions (WIS) by centrifugation. A discontinuous sucrose gradient was utilized to isolate non-sedimenting WIS fractions at the 25/45, 45/50, and 50+pellet interfaces. SDS-PAGE of these fractions exhibited filensin bands at approximately 115 and 53kDa based on previous literature. Western blot analysis was used to confirm filensin immunoreactivity. Gel bands were cut, digested with trypsin, and analyzed by HPLC tandem mass spectrometry (LC/MS/MS).
The molecular structure of filensin present in the 115kDa and the 53kDa bands was characterized via MALDI-MS and LC/MS/MS. At least 74% of the entire filensin amino acid sequence was covered in the 115 kDa sample using this proteomics approach. In addition, three sites of phosphorylation were detected on C-terminal serine residues. Putative truncation sites were also identified.
Filensin enrichment combined with proteomics analysis provided extensive sequence coverage and allowed characterization of filensin modifications including phosphorylation and truncation sites. These data will be used in future experiments to determine age-related modifications and in molecular crosslinking experiments between filensin and aquaporin 0 to determine specific sites of interaction.
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