No abstract

A two-dimensional (2D) HPLC-MS/MS method has been established for the determination of trace amounts of D-amino acid residues in proteins. D-Amino acid residues are now increasingly recognized as biomarkers of diseases and key moieties for the regulation of protein structures/functions, therefore, a sensitive analytical method is highly required. However, non-negligible amounts of D-amino acids are produced by chemical racemization during the hydrolysis of proteins, and a sensitive determination of D-amino acid residues is normally difficult. In the present study, DCl/D 2 O hydrolysis is adopted and the produced deuterated D-amino acids are distinguished from naturally-occurring D-amino acids in the proteins using the 2D-HPLC-MS/MS system. D-Ala, D-Asp, D-Glu, D-Pro and D-Ser (frequently observed D-amino acids in higher animals) were selected as the targets, and the sensitive determination (around 1% or less of the L-forms) could be carried out. As an application, the D-amino acid residues in ovalbumin (OVA) were determined, and the presence of a significant amount of D-Ser (1.8% of L-Ser) was demonstrated.

A new peak identification/quantification method, i.e., "shape-fitting" method, has been devised and applied to the determination of chiral amino acids in human plasma and urine. The shape fitting method enables the determination of target analytes using a part of the peak by mathematically predicting the whole peak even when the baseline is not clear. Using this method, D-glutamic acid (Glu) and D-proline (Pro) as well as their L-enantiomers in the plasma and urine were determined. The calibration lines of 4 target enantiomers were linear with correlation coefficients higher than 0.9998; the RSD values of the intra-day precision and inter-day precision were less than 6.70%. In the human plasma, trace amount of D-Pro (0.47 nmol/mL) was observed, and in the urine, 2.97 nmol/mL of D-Glu and 0.08 nmol/mL of D-Pro were successfully determined.

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 ( 2 HCl/ 2 H 2 O) 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, NH 2 -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.