Characterization of glycated hemoglobin in diabetic patients: usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation

A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated h emoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glyc ation by electrospray ionization mass spectrometry (ES-MS) of intact globin s 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 s ites within a and p globins was developed, e.g., Glu-C/Asp-N proteolytic di gestion followed by LC-ES-MS. Relative site occupancy within discrete compo nents of GHb was evaluated. A correlation between the degree of glycation m easured at Hb level (by affinity chromatography) and at globin level (measu red by ES-MS) was carried out. The above studies led us to conclude that du ring the process of phenylboronate chromatography GHb dimers, rather than t etramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of g lycation affects a much higher number of native Hb tetramers than was previ ously contemplated. No glycation sites appear to be missed by phenylboronat e affinity chromatography. We have found no evidence of the presence of mul tiple glycations within a single globin chain. While glycation of both glob ins within a dimer cannot be excluded, it is unlikely to be a significant p henomenon. According to ES-MS data, an equivalent of about one globin per c rp dimer of the affinity chromatography-isolated GHb carried glycation. (C) 2001 Elsevier Science B.V. All rights reserved.