title: Identification and relative quantification of specific glycation sites in human serum albumin.
abstract: Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys(549) (K(Am(Glc))QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.
authors.affiliation: Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Andrej
authors.initials: A
authors.lastname: Frolov
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
mesh_terms: D001665:Binding Sites; D003924:Diabetes Mellitus, Type 2; D017127:Glycation End Products, Advanced; D006031:Glycosylation; D006801:Humans; D008239:Lysine / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D017127:Glycation End Products, Advanced; D012709:Serum Albumin; D008239:Lysine