MALDI tissue imaging of ocular lens alpha-crystallin.
OBJECTIVE
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-microm 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-microm 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. alphaA-crystallin showed extensive truncation across whole sections, especially in the nuclei, whereas alphaB-crystallin was observed to be relatively stable. Both alphaA-crystallin and alphaB-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.
Han
Jun
J
Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Schey
Kevin L
KL
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D007908:Lens, Crystalline / Q000378:metabolism*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D038202:alpha-Crystallin A Chain / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000378:metabolism*
D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain