This laboratory has used a proteomics approach to study the proteins of the normal human lens and in cataracts of various etiologies. Lens dissections were done to obtain age-defined populations of fiber cells from lenses of all ages. Methodologies have been developed to yield high resolution separation of these proteins using two-dimensional polyacrylamide gel electrophoresis. A major endeavor of the lab is the identification of post-translational modifications of proteins, a major mechanism for regulating protein function, location in the cell and the association with other proteins. Identification of the proteins and the characterization of their post-translational modifications are done by matrix assisted laser desorption ionization time of flight and electrospray mass spectrometry. Previous results from this laboratory have demonstrated extensive post-translational modifications of the crystallins that begin early in life. These are spatially and temporally controlled, are necessary for the development of the normal adult human lens nucleus, and are certain to determine and alter crystalline function in different regions of the normal lens. Quantitative changes in some of the modified crystallins and additional modifications of other crystallins are found in pathology. These are being defined for alpha- and beta-crystallins. The most prominent modifications include C-terminal cleavages, deamidation, phosphorylation, and methionine oxidation. The results include several previously unreported modification sites. MIP/AQP0 is the most abundant membrane protein in the human lens and is thought to have a role in water permeability. Changes in structure/function of this membrane protein may be critical in mechanisms of pathology. The post-translational modifications of this protein have been temporally and spatially defined in the human lens. The major modifications identified, phosphorylation, deamidation and truncation of the polypeptide chain, may have critical functional significance.
