title: Identification of the site of glycation of human insulin.
abstract: This study evaluates the nature of glycated human insulin formed following exposure to hyperglycemic conditions in vitro. Glycated insulin was purified by RP-HPLC and its molecular mass (5971.3 Da) determined by plasma desorption mass spectrometry (MS). The difference in mass (163.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single reduced glucose (glucitol) residue. Following reduction of insulin disulfide bridges, MS confirmed that the B-chain was glycated. Enzymatic digestions with trypsin, endoproteinase Glu-C, and thermolysin, followed by RP-HPLC and identification of fragments by MS, localized glycation to the B-chain (1-5) region. Electrospray tandem MS identified the site of glycation as the B-chain NH2-terminal Phe1 residue. This was confirmed by automated Edman degradation with glycated human insulin.
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, N. Ireland, UK.
authors.forename: F P
authors.initials: FP
authors.lastname: O'Harte
authors.forename: P
authors.initials: P
authors.lastname: Højrup
authors.forename: C R
authors.initials: CR
authors.lastname: Barnett
authors.forename: P R
authors.initials: PR
authors.lastname: Flatt
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation
chemical_list: D007328:Insulin; D010446:Peptide Fragments; C039522:insulin, glycosylated