MALDI tissue Imaging of ocular lens α-crystallin

PURPOSE. To apply MALDI (matrix-assisted laser desorption ionization) tissue imaging methods to obtaining a profile of the distribution of the lens alpha-crystallins and their modified forms in calf and mature bovine lenses. METHODS. Frozen bovine lenses were cut equatorially at -12 degrees C to -20 degrees C into 10- to 40-mu m 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 alpha-crystallin and their modifications over approximately one-half of a single tissue section were reconstituted from mass spectral data sets acquired in 250-mu m 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 alpha-crystallin and their modified forms. alpha A-crystallin showed extensive truncation across whole sections, especially in the nuclei, whereas alpha B-crystallin was observed to be relatively stable. Both alpha A-crystallin and alpha 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 alpha-crystallin revealed in this study provide new leads in the investigation of underlying physiological significance of the modified forms of the two alpha-crystallin subunits.