Age-related changes in the spatial distribution of human lens alpha-crystallin products by MALDI imaging mass spectrometry.

Grey AC, Schey KL
Invest Ophthalmol Vis Sci. 2009 50 (9): 4319-29

PMID: 19387068 · PMCID: PMC2760347 · DOI:10.1167/iovs.09-3522

PURPOSE - To develop a protocol for MALDI (matrix-assisted laser desorption ionization) imaging mass spectrometry for mapping the distributions of alpha-crystallin and its modified forms in human lens tissue as a function of lens age and cataract.

METHODS - Frozen human lenses were cryosectioned equatorially and axially into 20-mum-thick sections, and the sections were mounted onto conductive glass slides by methanol soft-landing. An ethanol washing procedure facilitated uniform matrix crystal formation by a two-step matrix deposition procedure to produce high-quality mass spectral data. Molecular images of modified and unmodified alpha-crystallin subunits were obtained from mass spectral data acquired in 100-mum steps across normal and cataractous lens sections. Proteins extracted from the lens sections were digested with endoproteinase Glu-C and subjected to mass spectrometric analysis for identification of modifications.

RESULTS - Intact alpha-crystallin signals were detected primarily in the outer cortical fiber cells in lenses up to 29 years of age. Multiple truncation products were observed for alpha-crystallin that increased in abundance, both with distance into the lens and with lens age. Phosphorylated alphaB-crystallin forms were most abundant in the cortical region of older lenses. In axial sections, no significant anterior-posterior pole variation was observed. A previously unreported alphaA-crystallin mutation was detected in an age-matched cataractous human lens.

CONCLUSIONS - A method has been developed to spatially map the age-related changes of human lens alpha-crystallin by MALDI imaging mass spectrometry including a novel L52F alphaA-crystallin mutation in a cataractous lens. Application of this spatially resolved proteomic technique to lens biology enhances the understanding of alpha-crystallin protein processing in aging and diseased human lenses.

MeSH Terms (14)

Adult Aged Aging alpha-Crystallin A Chain alpha-Crystallin B Chain Blotting, Western Cataract Child Electrophoresis, Polyacrylamide Gel Humans Lens, Crystalline Middle Aged Protein Processing, Post-Translational Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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