A rapid and sensitive detection of D-Aspartic acid in Crystallin by chiral derivatized liquid chromatography mass spectrometry

作者：Mizuno Hajime; Miyazaki Yasuto; Ito Keisuke; Todoroki Kenichiro; Min Jun Zhe; Toyo'oka Toshimasa^*

来源：Journal of Chromatography A, 2016, 1467: 318-325.

DOI：10.1016/j.chroma.2016.07.021

• Crystallin
• D-Asp
• DBD-Py-NCS
• ADME column separation
• Chiral protoeomics
• LC-MS

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 alpha-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 alpha-Crystallin standard and UV irradiated alpha-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 alpha A- and alpha B-Crystallin homologous subunits of alpha-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 alpha-Crystallin, AsP76 and Asp(84) in the alpha A-Crystallin and Asp(96) in alpha B-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.

• 出版日期2016-10-7