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C. O. Asomugha, R. Gupta, O. P. Srivastava; In situ Laser Capture Microdissection Regional Analysis of Crystallin Distribution in Human Lens Tissue. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4091.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the regional distribution of crystallins and their post-translationally modified species, in situ, in aging human lenses using the laser capture microdissection (LCM) method.
Human lenses were excised from globes and stored at -80° C until used. Lenses were brought to room temperature, embedded directly in optimum cutting temperature (O.C.T.) medium, and frozen inside of a cryostat chamber. Fifteen micrometer sections of the lens were mounted on polyethylene naphthalate membrane slides. Outer cortical, inner cortical and nuclear regions of a normal lens from a 65-year old donor were selectively captured and catapulted onto separate microfuge caps. Captured tissue from each region was solubilized in 30 µL of Buffer A (50 mM TRIS, 1 mM PMSF, 1 mM Iodoacetamide, 1 mM DTT). Equal amounts of protein from the three regions were analyzed by SDS-PAGE. Individual protein bands were excised from a SDS-PAGE gel, trypsin-digested, and tryptic fragments were analyzed by the MALDI-TOF mass spectrometric method.
SDS-PAGE analysis showed an average of 7 - 10 protein bands in each of the three lenticular regions. MALDI-TOF analysis showed αA, αB, βB1, βB2, and fragments of γS-crystallin in the outer cortical region. The inner cortical and nuclear regions contained βA3, βB1, γD, and γS crystallins, but, surprisingly αA and αB were not detected for yet unknown reasons. βA3 crystallin was present in both the inner cortex and the nucleus, but showed varying molecular weights.
MALDI-TOF analysis showed the presence of αA and αB crystallins in the outer cortex and βA3 in both inner cortical and nuclear regions of the lens. However, a truncated βA3 crystallin species was found in the nuclear region.
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