Age-related changes in the spatial distribution of human lens alpha-crystallin products by MALDI imaging mass spectrometry.
OBJECTIVE
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.
Grey
Angus C
AC
Department of Biochemistry, Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232-8575, USA.
Schey
Kevin L
KL
D000328:Adult; D000368:Aged; D000375:Aging / Q000502:physiology*; D015153:Blotting, Western; D002386:Cataract / Q000378:metabolism*; D002648:Child; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D008875:Middle Aged; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D038202:alpha-Crystallin A Chain / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000378:metabolism*
D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain