Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?
We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.
Wilmarth
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Department of Integrative Biosciences, School of Dentistry, Oregon Health and Science University, 611 South West Campus Drive, Portland, Oregon 97239, USA. wilmarth@ohsu.edu
Tanner
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Dasari
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Nagalla
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Riviere
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Bafna
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Pevzner
P A
PA
David
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D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D000577:Amides / Q000032:analysis*; D000595:Amino Acid Sequence; D003459:Crystallins / Q000032:analysis*; D003545:Cysteine / Q000032:analysis; D006801:Humans; D007231:Infant, Newborn; D007908:Lens, Crystalline / Q000737:chemistry*; D008297:Male; D008745:Methylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D012995:Solubility
D000577:Amides; D003459:Crystallins; D010455:Peptides; D003545:Cysteine