Age-Related Changes in the Spatial Distribution of Human Lens α-Crystallin Products by MALDI Imaging Mass Spectrometry

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-mu m-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-mu m 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 alpha B-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 alpha A-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 alpha A-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. (Invest Ophthalmol Vis Sci. 2009;50:4319-4329) DOI: 10.1167/iovs.09-3522