학술논문

Two-Dimensional HPLC-MS/MS Determination of Multiple D-Amino Acid Residues in the Proteins Stored Under Various pH Conditions
Document Type
Journal Article
Source
CHROMATOGRAPHY. 2017, 38(2):65
Subject
2D-HPLC MS/MS
D-Amino acid residue
Enantiomer separation
Isomerization
Language
English
ISSN
1342-8284
1348-3315
Abstract
The formation of D-amino acid residues in proteins is considered as one of the deterioration processes, and the determination of these D-amino acid residues is highly expected for the screening of new biomarkers under various disease conditions. In the present study, a two-dimensional (2D) HPLC-MS/MS system following the hydrolysis with deuterium chloride (2HCl/2H2O) and derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been designed/developed and applied to the analysis of proteins stored under various conditions. As the target, 5 major D-amino acid residues (Ala, Asp, Glu, Pro and Ser) were selected. The analytical procedure was validated using a model peptide, NH2-Gly-Pro-Glu-Ala-Asp-Ser-Gly-OH, and the obtained calibration lines of %D for the 5 target amino acids were linear with correlation coefficients greater than 0.998. The RSD values for the intra-day precision and inter-day precision were lower than 5%. In most of the proteins tested, the amounts of the D-Ser and D-Asp residues increased during storage, and the highest value (14%, D-Ser) was observed in ovalbumin (OVA) after storage at pH 9.5 for 4 weeks.