Enantioselective Determination of Alanine, Aspartic Acid and Serine Residues in Proteins Stored Under High Temperature and Basic pH Conditions

d-Amino acid residues have been observed in aged proteins in various diseases, and the correlations between the formation of d-form residues and functional changes are of increasing interest. As the factors causing the epimerization of amino acids residues, external stresses, such as high temperature and alkaline conditions, have been reported, and there is a pressing need to clarify the production of d-form residues in proteins exposed to various stress conditions. In the present study, d-amino acid residues of alanine (Ala), aspartic acid (Asp) and serine (Ser) were determined in proteins stored under high temperature and/or basic pH conditions. The protein samples were hydrolyzed, then the obtained amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and analyzed by using a two-dimensional chiral LC-MS/MS system equipped with Singularity RP18 (first dimension) and Singularity CSP-013S (second dimension) columns. As a result, the d-Ser residue (3.5 %) was detected in ovalbumin (OVA) stored at 37 ºC and pH 7.4 for 4 weeks (physiological conditions), while relatively large amounts of d-Asp (5.4 %) and d-Ser (9.4 %) residues were observed in the same protein stored at 60 ºC and pH 7.4 for 4 weeks. In OVA stored at 60 ºC and pH 9.6 for 4 weeks, large amounts of d-Asp and d-Ser residues (21.9 % and 37.8 %) were determined, while a non-negligible amount of d-Ala residues was observed (2.9 %). Also, in human serum albumin stored at 60 ºC and pH 9.6 for 4 weeks, the d-forms of all target amino acid residues were observed and the %d values were 2.3 for Ala, 15.3 for Asp and 33.5 for Ser. These results indicated that epimerization of various amino acid residues occurs (not only for Asp and Ser) by the external stress and further detailed studies are expected.