Characterization of glycated hemoglobin in diabetic patients

Usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation

Xinyi Zhang, Katalin F. Medzihradszky, John Cunningham, Phillip Lee, Cheryl L. Rognerud, Ching Nan Ou, Paul Harmatz, H. Ewa Witkowska

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7% GHb. An efficient protocol for mapping glycation sites within α and β globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC-ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per αβ dimer of the affinity chromatography-isolated GHb carried glycation.

Original languageEnglish (US)
Pages (from-to)1-15
Number of pages15
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume759
Issue number1
DOIs
StatePublished - Aug 5 2001
Externally publishedYes

Fingerprint

Globins
Glycosylated Hemoglobin A
Mass spectrometry
Affinity chromatography
Electrospray ionization
Dimers
Monitoring
Hemoglobins
Chromatography
Structural analysis
Ion exchange
Resins
Molecules
benzeneboronic acid

Keywords

  • Glycated
  • Hemoglobin

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Characterization of glycated hemoglobin in diabetic patients : Usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation. / Zhang, Xinyi; Medzihradszky, Katalin F.; Cunningham, John; Lee, Phillip; Rognerud, Cheryl L.; Ou, Ching Nan; Harmatz, Paul; Witkowska, H. Ewa.

In: Journal of Chromatography B: Biomedical Sciences and Applications, Vol. 759, No. 1, 05.08.2001, p. 1-15.

Research output: Contribution to journalArticle

Zhang, Xinyi ; Medzihradszky, Katalin F. ; Cunningham, John ; Lee, Phillip ; Rognerud, Cheryl L. ; Ou, Ching Nan ; Harmatz, Paul ; Witkowska, H. Ewa. / Characterization of glycated hemoglobin in diabetic patients : Usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation. In: Journal of Chromatography B: Biomedical Sciences and Applications. 2001 ; Vol. 759, No. 1. pp. 1-15.
@article{ff1cd69938f94d4cb075caa1156911fe,
title = "Characterization of glycated hemoglobin in diabetic patients: Usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation",
abstract = "A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7{\%} GHb. An efficient protocol for mapping glycation sites within α and β globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC-ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per αβ dimer of the affinity chromatography-isolated GHb carried glycation.",
keywords = "Glycated, Hemoglobin",
author = "Xinyi Zhang and Medzihradszky, {Katalin F.} and John Cunningham and Phillip Lee and Rognerud, {Cheryl L.} and Ou, {Ching Nan} and Paul Harmatz and Witkowska, {H. Ewa}",
year = "2001",
month = "8",
day = "5",
doi = "10.1016/S0378-4347(01)00196-7",
language = "English (US)",
volume = "759",
pages = "1--15",
journal = "Journal of Chromatography B: Biomedical Sciences and Applications",
issn = "0378-4347",
publisher = "Elsevier BV",
number = "1",

}

TY - JOUR

T1 - Characterization of glycated hemoglobin in diabetic patients

T2 - Usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation

AU - Zhang, Xinyi

AU - Medzihradszky, Katalin F.

AU - Cunningham, John

AU - Lee, Phillip

AU - Rognerud, Cheryl L.

AU - Ou, Ching Nan

AU - Harmatz, Paul

AU - Witkowska, H. Ewa

PY - 2001/8/5

Y1 - 2001/8/5

N2 - A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7% GHb. An efficient protocol for mapping glycation sites within α and β globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC-ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per αβ dimer of the affinity chromatography-isolated GHb carried glycation.

AB - A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7% GHb. An efficient protocol for mapping glycation sites within α and β globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC-ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per αβ dimer of the affinity chromatography-isolated GHb carried glycation.

KW - Glycated

KW - Hemoglobin

UR - http://www.scopus.com/inward/record.url?scp=0035812225&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035812225&partnerID=8YFLogxK

U2 - 10.1016/S0378-4347(01)00196-7

DO - 10.1016/S0378-4347(01)00196-7

M3 - Article

VL - 759

SP - 1

EP - 15

JO - Journal of Chromatography B: Biomedical Sciences and Applications

JF - Journal of Chromatography B: Biomedical Sciences and Applications

SN - 0378-4347

IS - 1

ER -