Mass spectrometric analysis of human lens crystallins

The disease cataract, a major cause of blindness worldwide, is an opacification of the lens. Post-translational modifications which disrupt the close-packing of the crystallins are likely causes of the change in refractive index and the formation of opacities observed in cataractous lenses. This study is a comprehensive examination of the water-insoluble crystallins and water-soluble γ-crystallins from normal human lenses. The crystallins were isolated by a combination of chromatographic techniques. Electrospray ionization mass spectrometry was used to identify the crystallins and their post-translational modifications. The in vivo modifications identified in this investigation will help to form a basis of differentiation between normal and cataractous lens crystallins and provide insight into the possible causes of cataract formation. Water-soluble γ-crystallins were isolated by size exclusion and reversed phase chromatography. The molecular weights of the intact proteins were determined by electrospray ionization mass spectrometry. This investigation has demonstrated that only, three γ-crystallins, γS, γD and γC, are present in the water-soluble lens portion. Mass spectrometric analysis of the peptides produced by enzymatic digestions of the intact proteins was performed. Post-translational modifications were indicated by molecular weights of peptides that differed from the he calculated molecular weights. The specific sites of modification were determined. The major post-translational modifications of the γ-crystallins, were identified as deamidation and disulfide bonding and were both found to increase with age. Water-insoluble crystallins were isolated after incubation in 6M guanidine-hydrochloride for one week by size exclusion chromatography. Further fractionation of the water-insoluble crystallins was performed using a polystyrene reversed phase column and a temperature of 50°\u27C. Molecular weights of the intact proteins were determined by electrospray ionization mass spectrometry. The proteins identified in this study included αA, αB, βB2, γS and γD and were all found intact and without modifications altering their molecular weight by more than 5 Da. Enzymatic digestion of the water-insoluble fractions provided evidence for deamidation, disulfide bonding and degradation of βB1-, αA- and γS-crystallins