title: Quantification of glycated N-terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry.
abstract: A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1-bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC-MS/MS. In this study, 1-deoxyfructosyl-VHLTPE and VHLTPE, which are N-terminal peptides of the β-chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1-bromobutane and analyzed with LC-MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non-glycated peptide.
authors.affiliation: Bio-Medical Standard Section, National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, Tsukuba C-3, Ibaraki, 305-8563, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Sakaguchi
authors.affiliation: Bio-Medical Standard Section, National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, Tsukuba C-3, Ibaraki, 305-8563, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kinumi
authors.affiliation: Bio-Medical Standard Section, National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, Tsukuba C-3, Ibaraki, 305-8563, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Bio-Medical Standard Section, National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, Tsukuba C-3, Ibaraki, 305-8563, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Takatsu
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006031:Glycosylation; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000596:Amino Acids; D006454:Hemoglobins
keywords: LC-MS/MS; amino acid analysis; derivatization; glycated amino acid; isotope dilution MS