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

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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 (R-P-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). (C) 2002 Elsevier Science B.V. All rights reserved.
LanguageEnglish
Pages1-8
JournalRegulatory Peptides
Volume113
Issue number1-3
DOIs
Publication statusPublished - May 2003

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Proinsulin
Electrospray ionization
Mass spectrometry
Ions
Sorbitol
Disulfides
Peptides
Dithiothreitol
High performance liquid chromatography
Insulin

Cite this

@article{0e5486cfc6754eaa882b736712802144,
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 (R-P-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). (C) 2002 Elsevier Science B.V. All rights reserved.",
author = "Aine McKillop and A Meade and Peter Flatt and Finbarr O'Harte",
year = "2003",
month = "5",
doi = "10.1016/S0167-0115(02)00292-6",
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T1 - Evaluation of the site(s) of glycation in human proinsulin by ion-trap LCQ electrospray ionization mass spectrometry

AU - McKillop, Aine

AU - Meade, A

AU - Flatt, Peter

AU - O'Harte, Finbarr

PY - 2003/5

Y1 - 2003/5

N2 - 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 (R-P-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). (C) 2002 Elsevier Science B.V. All rights reserved.

AB - 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 (R-P-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). (C) 2002 Elsevier Science B.V. All rights reserved.

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DO - 10.1016/S0167-0115(02)00292-6

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