Differentiation and Semiquantitative Analysis of an Isoaspartic Acid in Human α-Crystallin by Postsource Decay in a Curved Field Reflectron
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- 作者：Yuzo Yamazaki ; Norihiko Fujii ; Yutaka Sadakane ; Noriko Fujii
- 刊名：Analytical Chemistry
- 出版年：2010
- 出版时间：August 1, 2010
- 年：2010
- 卷：82
- 期：15
- 页码：6384-6394
- 全文大小：399K
- 年卷期：v.82,no.15(August 1, 2010)
- ISSN：1520-6882
文摘
α-Crystallin, which forms a huge multimeric complex that is essential for maintaining eye lens transparency, is one of the major proteins in the lens. The protein, which exists as isoforms αA and αB, functions as a molecular chaperone to restore the original conformations of distorted constituent proteins in the lens. This function is important to maintain the transparency of the lens, because there is no protein turnover in the lens. Abnormal aggregation of constituent proteins in the lens has been reported in cataract patients, and deamidation of Asn as well as racemization and isomerization of Asp have been found in the α-Crystallin of these patients. While the establishment of a quick and facile analytical method is eagerly anticipated to investigate the relevance of the isomerization to pathological states such as cataracts, differentiating the isomerization states is still not performed routinely. Here, we report the differentiation and semiquantitative analysis of an isoaspartic acid (βAsp) in human α-Crystallin using postsource decay on a MALDI-TOF mass spectrometer incorporating a curved field reflectron. Our reproducible results of analyzing synthetic and tryptic peptides containing βAsp corroborated results obtained using a previously reported diagnostic ion, − 46, for the differentiation of βAsp. The relative content of βAsp in the peptide was successfully estimated from a unique ratio, , corresponding to cleavages at the C- and N-termini, respectively, of the isomeric residues. The βAsp content was consistent with measurements obtained independently by reversed phase HPLC analysis. Experiments in which neighboring amino acids adjacent to βAsp/Asp were substituted revealed that the ratio between and reflected the isomerization status, while the diagnostic ion was observed only in the peptides that included an arginine residue at their C-terminus. Postsource decay experiments utilizing both the diagnostic ion and the characteristic fragment pattern could be applied to various kinds of peptides containing βAsp.