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K Srivastava, OP Srivastava; Determination of Modifications in Three Isoforms of a 9 kDa Gamma D-Crystallin Fragment Isolated from Human Lenses . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4659.
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Purpose: The aim of the study was to characterize post-translational modifications in the three isoforms of a 9 kDa γD-crystallin fragment. Method: The three isoforms of a 9 kDa γD-crystallin fragment were isolated as previously described (Exp. Eye Res.62, 593-604, 1996), and were further separated by two-dimensional gel elctrophoresis. Individual isoforms were excised from 2D-gels and digested with either trypsin or chymotrypsin and analyzed by mass spectrometry. Results: By mass spectrometric analysis, the 9 kDa isoforms I, II and III showed Mr of 11033, 10903 and 10868, respectively. On chymotrysin digestion followed by mass spectrometric analyses of the isoforms, a fragment with a mass of 1374 (representing residue no. 154 to 166 of γD with a sequence of QDWGATNARVGSL) was detected in the isoform III but was at very low levels or almost non-existent in the isoforms I and II. A similar mass spectrometric analysis following trysin digestion of the isoforms, showed that W at position 156 was modified and M at position 146 was oxidized. Conclusion: The results show a potential modification region of residue no. 154-166 in the three isoforms of γD-crystallin fragment in human lenses. Two residues, M and W at positions 146 and 156, respectively, were modified in all the three isoforms.
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