Quantitation of asparagine-101 deamidation from alpha-A crystallin during aging of the human lens

PURPOSE: To quantitate deamidation of asparagine-101 from the alpha-A crystallin protein of human lenses of different ages. METHODS: Alpha-A crystallin was purified from total proteins of human lenses of different ages, followed by tryptic digestion and resolution of the peptides, using reverse phase chromatography. Known amounts of synthetic peptide standards, corresponding to the amidated and deamidated forms of the expected tryptic peptide containing asparagine-101, were used to identify and quantitate the amount of deamidation. RESULTS: From 0-30 yrs of age, approximately 45% of asparagine-101 was deamidated, while only approximately 5% additional deamidation occurred during 30-68 yrs of age. CONCLUSIONS: In the normal human lens, most deamidation of asparagine-101 occurs during the first approximately 30 years of age, followed by a small additional amount of deamidation (approximately 5%) during the next approximately 38 years, resulting in a maximum of approximately 50% deamidation during the lifetime of the individual.