CHROMATOGRAPHY
Online ISSN : 1348-3315
Print ISSN : 1342-8284
Original papers
Two-Dimensional HPLC-MS/MS Determination of Multiple D-Amino Acid Residues in the Proteins Stored Under Various pH Conditions
Chiharu ISHIITetsuya MIYAMOTOShoto ISHIGOYurika MIYOSHIMasashi MITAHiroshi HOMMATadashi UEDAKenji HAMASE
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2017 Volume 38 Issue 2 Pages 65-72

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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.

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© 2017 The Society for Chromatographic Sciences
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