Purpose

To develop a general tissue preparation protocol for MALDI (Matrix-Assisted Laser Desorption Ionization) imaging mass spectrometry of ocular lens tissue, and to compare the spatial distributions of α-crystallin and its modified forms in bovine and rabbit lenses.

Methods

Frozen bovine and rabbit lenses were cryosectioned equatorially at− 20 C into 12 μm-thick tissue sections. Lens sections were mounted onto conductive glass slides by ethanol soft-landing to maintain tissue integrity. An ethanol/xylene washing procedure was applied to each section before matrix application to facilitate uniform matrix crystal formation across the entire tissue section. Molecular images of both α-crystallin subunits and their modified forms were obtained from mass spectral data acquired at 100 μm steps across both whole rabbit and half bovine lens sections.

Results

Distinct spatial patterns for the two subunits of α-crystallin and their modified forms were observed in the rabbit and bovine lens sections. While αA-crystallin was extensively degraded in the lens core of both species, rabbit lenses exhibited a greater degree of larger molecular weight truncation products. In contrast, αB-crystallin degradation was limited in both species. Interestingly, phosphorylation of αA-and αB-crystallin was most abundant in the middle cortex of both species.

Conclusions

An improved method for investigating the spatial distribution of α-crystallin in the ocular lens by MALDI imaging mass spectrometry has been developed. The localization of multiple degradation products and specific regions of α-crystallin phosphorylation in bovine and rabbit lenses gives new insight into the program of lens fiber cell differentiation and normal lens function.