Jun Han and Kevin L. Schey
Investigative Ophthalmology and Visual Science. 2006, 47, pp 2990-2996

PURPOSE. To apply MALDI (matrix-assisted laser desorption ionization) tissue imaging methods to obtaining a profile of the distribution of the lens -crystallins and their modified forms in calf and mature bovine lenses.

METHODS. Frozen bovine lenses were cut equatorially at –12 °C to –20°C into 10- to 40-µm sections depending on lens age. Tissue sections were mounted onto MALDI sample plates by ethanol soft-landing to maintain tissue integrity. A two-layered matrix deposition method was used to improve mass spectral reproducibility across sections. Molecular images of the two subunits of -crystallin and their modifications over approximately one-half of a single tissue section were reconstituted from mass spectral data sets acquired in 250-µm steps. Identification of protein truncation products and confirmation of phosphorylation distribution patterns were performed by reverse-phase liquid chromatography of soluble extracts from specific tissue regions followed by tandem mass spectrometry (LC/MS/MS).

RESULTS. Distinct distribution patterns were observed for the two subunits of -crystallin and their modified forms. A-crystallin showed extensive truncation across whole sections, especially in the nuclei, whereas B-crystallin was observed to be relatively stable. Both A-crystallin and B-crystallin displayed the highest level of phosphorylation in the middle cortex region, a finding confirmed by LC/MS/MS analysis of dissected regions.

CONCLUSIONS. A new imaging technique has been successfully applied to molecularly characterize the spatial distribution of lens proteins and their modifications in lens sections. The different distributions of -crystallin revealed in this study provide new leads in the investigation of underlying physiological significance of the modified forms of the two -crystallin subunits.

DOI:  10.1167/iovs.05-1529



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