Development of a stable isotope dilution assay for an accurate quantification of protein-bound N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine using a (13)C-labeled internal standard.

摘要

Syntheses of the labeled Amadori compound [(13)C(6)]-N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine ([(13)C(6)]-DFLys) and the labeled glycated tetrapeptide Ala-[(13)C(6)]-DFLys-Leu-Gly are presented. The compounds were used in the development of stable isotope dilution assays for the quantification of the degree of glycosylation of bovine serum albumin treated for 20 min at 95 degrees C in the presence of glucose. The experiments revealed that the use of the labeled standards in combination with LC/MS allowed the exact quantification of protein-bound DFLys with the high recovery rate of 95% (at a spike level of 150 nmol/mg of protein) and a low detection limit of 5 nmol/mg of protein. The data revealed, however, that DFLys is significantly degraded during the enzymic hydrolysis of the protein backbone generally needed in the quantification procedure and, furthermore, incomplete digestion of the protein was observed. Both sources of errors were clearly overcome by using in particular the labeled peptide as the internal standard.