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J. Han, K. Schey; Molecular Tissue Imaging of Lens Protein Modification . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3879.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: This work is focused on development of MALDI imaging methods to detect ocular lens proteins and their modifications with spatial resolution. Methods: Frozen bovine lens tissue was sectioned into 12µm thick slices in a cryostat and placed directly onto a MALDI sample plate. After application of organic acid and deposition of sinapinic acid matrix, lens tissue was subjected to MALDI/MS on a DE–STR time–of–flight mass spectrometer. Images of the major lens proteins and their degradation products during cell maturation were acquired. Identification of the protein degradation products was accomplished based on "top down" proteomics with capillary LC separation of soluble intact proteins and tandem mass spectrometry. Results: Direct MALDI imaging of sectioned lens tissue showed distinct protein expression patterns in the specific lens regions. Significantly increased protein signals under MW 20,000 were observed in the nucleus than in the equatorial cortex due to protein degradation during fiber cell maturation. Most of the degradation products identified by the top down approach were from the lens structural proteins, such as αA– and αB–crystallins. Over 30 degradation products were observed and identified in a single nuclear section. Conclusions: Molecular tissue imaging of the ocular lens proteins provides a means to visually view lens protein expression and degradation profiles with spatial resolution. The complementary "top down" strategy was successfully used as a method for identifying protein degradation in ocular lens. Further refinement of these methodologies is underway to improve sensitivity and spatial resolution.
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