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MALDI Tissue Imaging of Ocular Lens -Crystallin

From the Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina.

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.

This article has been cited by other articles:

				P. Santhoshkumar, P. Udupa, R. Murugesan, and K. K. Sharma Significance of Interactions of Low Molecular Weight Crystallin Fragments in Lens Aging and Cataract Formation J. Biol. Chem., March 28, 2008; 283(13): 8477 - 8485. [Abstract] [Full Text] [PDF]