Abstract
Abstract: :
Purpose: To determine by mass spectrometry the number and mass of the protein modifications present in enzymatic digests of Type I–II Indian cataract lens proteins and to compare them to the modifications formed by ascorbic acid glycation of lens proteins in vitro. Methods: The water–insoluble proteins from Type I – II Indian cataract lenses were pooled and digested with a battery of proteolytic enzymes to release the modified amino acids. BioGel P–2 size exclusion chromatography was carried out and the peaks of "yellow" amino acids were collected based upon their absorbance at 330 nm. Each of these peaks was subjected to HPLC/MS and the mass elution maps determined. The samples were again analyzed and the significant mass peaks (∼106 response factor) were subjected to MS/MS analysis to identify the daughter ions of each modification. Calf lens proteins were reacted with 20 mM ascorbic acid in air for 4 weeks, dialyzed and similarly digested with proteolytic enzymes. Mass spectrometric analyses of the digests of these proteins were conducted in the same manner. Results: BioGel P–2 chromatography separated each digest into four A330nm–absorbing peaks: an early peak (peak 1), which likely also contained several undigested peptides, peaks 2 and 3, which were the major peaks and which co–eluted with the amino acid fraction and peak 4, which contained aromatic amino acids. Mass spectrometric maps of peaks 1, 2 and 3 from aged human lenses showed 58, 40 and 55 mass values respectively, ranging from 200 to 600 amu. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values respectively with similar amu. A total of 43 species from each digest were analyzed by HPLC/MS/MS and 41 were shown to be identical. Unmodified calf lens proteins showed many similar mass values, but the response factors were 100 to 1000–fold less for every modification. Conclusions: Greater than 90% of the major modified amino acids present in Type I – II Indian cataract lens proteins appear to arise as a result of ascorbic acid modification, and are presumably advanced glycation endproducts. A glycomic analysis in this manner can be used to obtain a complete picture of the Maillard reaction products obtained with different carbonyl compounds, as well as the protein modifications that accumulate during aging.
Keywords: cataract • protein modifications–post translational • aging