Establishment of a Two-Dimensional HPLC-MS/MS Method Combined with DCl/D<sub>2</sub>O Hydrolysis for the Determination of Trace Amounts of D-Amino Acid Residues in Proteins

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/D2O 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.