title: Evaluation of the site(s) of glycation in human proinsulin by ion-trap LCQ electrospray ionization mass spectrometry.
abstract: The glycation of beta cell proteins is known to occur under hyperglycemic states. The site(s) of glycation in human proinsulin was investigated following exposure to a hyperglycemic environment under reducing conditions in vitro. Proinsulin and glycated proinsulin were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and identified using LCQ ion-trap electrospray ionization mass spectrometry. This revealed a major peak (>70% total) of monoglycated proinsulin (M(r) 9552.2 Da), a second peak (approximately 27%) of nonglycated proinsulin (M(r) 9389.8 Da), and a third minor peptide peak (approximately 3%) corresponding to diglycated proinsulin (M(r) 9717.9 Da). Following reduction of disulphide bridges with dithiothreitol, intact peptides were incubated with endoproteinase Glu-C to release nine daughter fragments for LC-MS analysis. This strategy revealed an N-terminal fragment of monoglycated proinsulin Phe(1)-Glu(13), which contained a single glucitol adduct (M(r) 1642.0 Da). A similar treatment of small amounts of purified diglycated proinsulin revealed a fragment with Phe(1)-Glu(13) linked by a disulphide bridge to Gln(70)-Glu(82) containing two glucitol adducts (M(r) 3292.7 Da). In summary, these studies indicate that the major site of glycation in proinsulin, like insulin, is the amino terminal Phe(1) residue. However, small amounts of diglycated proinsulin occur naturally, involving an additional site of glycation located between Gln(70) and Glu(82).
authors.affiliation: School of Biomedical Sciences, University of Ulster, Northern Ireland BT52 1SA, Coleraine, UK. am.mckillop@ulster.ac.uk
authors.forename: Aine M
authors.initials: AM
authors.lastname: McKillop
authors.forename: Angela
authors.initials: A
authors.lastname: Meade
authors.forename: Peter R
authors.initials: PR
authors.lastname: Flatt
authors.forename: Finbarr P M
authors.initials: FP
authors.lastname: O'Harte
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011384:Proinsulin / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013997:Time Factors
chemical_list: D010446:Peptide Fragments; D011384:Proinsulin