title: A rapid and sensitive detection of D-Aspartic acid in Crystallin by chiral derivatized liquid chromatography mass spectrometry.
abstract: A method for the determination of D-Aspartic acid (D-Asp) and its D/L ratio in peptides and proteins has been developed. This method was carried out with good separation of the D/L chiral peptide pairs by combination of a chiral derivatization and an ADME column separation. Furthermore, a cationic derivatization reagent, DBD-Py-NCS, increased the sensitivity of the ESI-MS/MS detection. To confirm the comprehensive peptide analysis, synthesized α-Crystallin tryptic peptides, which included D-Asp residues, were analyzed. The 5 pairs of D/L-Asp that included peptide diastereomers were well separated. Their peak resolutions were more than 1.5 and the results were reproducible (RSD<0.05, n=5). As an application of this method, we analyzed the α-Crystallin standard and UV irradiated α-Crystallin. After trypsin digestion and DBD-Py-NCS derivatization, the tryptic peptide derivatives were applied to LC-MS/MS. Based on the results of peptide sequence identification, almost all the tryptic peptides of the αA- and αB-Crystallin homologous subunits of α-Crystallin were detected as DBD-Py NCS derivatives. However, there was no D-Asp residue in the standard proteins. In the case of the UV irradiated α-Crystallin, Asp76and Asp84in the αA-Crystallin and Asp96in αB-Crystallin were racemized to D-Asp. These results show that this proposed chiral peptide LC-MS/MS method using chiral derivatization provides a rapid and sensitive analysis for post translational Asp racemization sites in aging proteins.
authors.affiliation: Laboratory of Analytical and Bioanalytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Mizuno
authors.affiliation: Laboratory of Analytical and Bioanalytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Miyazaki
authors.affiliation: Laboratory of Food Chemistry, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.affiliation: Laboratory of Analytical and Bioanalytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Todoroki
authors.affiliation: Laboratory of Analytical and Bioanalytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Jun Zhe
authors.initials: JZ
authors.lastname: Min
authors.affiliation: Laboratory of Analytical and Bioanalytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan. Electronic address: toyooka@u-shizuoka-ken.ac.jp.
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
mesh_terms: D002853:Chromatography, Liquid; D003459:Crystallins / Q000737:chemistry*; D026603:D-Aspartic Acid / Q000032:analysis* / Q000737:chemistry; D007202:Indicators and Reagents; D017879:Isothiocyanates / Q000737:chemistry; D010069:Oxadiazoles / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry
chemical_list: C107817:4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole; D003459:Crystallins; D007202:Indicators and Reagents; D017879:Isothiocyanates; D010069:Oxadiazoles; D010455:Peptides; D026603:D-Aspartic Acid
keywords: ADME column separation; Chiral protoeomics; Crystallin; D-Asp; DBD-Py-NCS; LC–MS