The proposed investigation will use new analytical methods based on mass spectrometry to identify human lens proteins and characterize their post-translational modifications. In the first level of analysis, two new types of mass spectrometry, electrospray ionization (ESIMS) and matrix-assisted laser desorption (MALDI), will be used to determine the molecular weights of intact proteins with an accuracy to 0.01%. In the second level of analysis, crystallins will be digested into peptides whose molecular weights will be determined by directly coupled high performance liquid chromatography mass spectrometry (HPLC/MS). Results for young, clear lenses will be used to make a data bank, to which results from aged and cataractous lenses will be compared. At present, reference data for alpha-crystallins from young and old clear lenses are nearly complete and considerable data have been collected for beta and gamma-crystallins.
The Specific Aims of the present proposal are 1). To complete the identification of beta- and gamma-crystallins that are expressed in the human lens and define post- translational modifications of these proteins associated with aging. 2). Identify the crystallin components and post-translational modifications of the water-insoluble, guanidine hydrochloride-soluble fraction of clear lenses from young and old donors. 3). Identify post-translational modifications of alpha-, beta-, and gamma-crystallins associated with early stages of cataract. 4). Through collaborative studies, identify the crystallin components and post-translational modifications of individual spots isolated from human lenses by 2-D gel electrophoresis.
Showing the most recent 10 out of 49 publications
