Evaluation of the site(s) of glycation in human proinsulin by ion-trap LCQ electrospray ionization mass spectrometry

• Published in: Regulatory peptides 2003-05, Vol.113 (1), p.1-8
• Main Authors: McKillop, Aine M., Meade, Angela, Flatt, Peter R., O'Harte, Finbarr P.M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Diverse techniques; Endoproteinase Glu-C; Fundamental and applied biological sciences. Psychology; Glycosylation; HPLC; Humans; Mass Spectrometry - methods; Molecular and cellular biology; Molecular Sequence Data; Molecular Structure; Nonenzymatic glycation; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Proinsulin; Proinsulin - chemistry; Proinsulin - metabolism; Protein Conformation; Time Factors
• ISSN: 0167-0115; 1873-1686
• DOI: 10.1016/S0167-0115(02)00292-6

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.