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 D.K. Lee, Cheryl L. Rognerud, Ching Nan Ou, Paul Harmatz, H. Ewa Witkowska

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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
Issue number1
StatePublished - Aug 5 2001
Externally publishedYes



  • Glycated
  • Hemoglobin

ASJC Scopus subject areas

  • Chemistry(all)

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