Structural analysis of glycated human hemoglobin using native mass spectrometry

Abstract

Glycated hemoglobin (GHb) is the indicator of the long-term glycemic index of an individual. GHb is formed by the irreversible modification of N-terminal α-amino group of β globin chain with glucose via Amadori rearrangement. Cation exchange chromatography exploits the difference in surface charges between GHb and native hemoglobin (HbA0) for their separation and quantification. However, glucose condensation is specific to primary amino groups. Therefore, structural characterization of GHb synthesized in vivo is essential as multiple glycation may interfere with GHb assessment. The stoichiometric composition of different glycated hemoglobin from a 19% GHb sample was deduced using native mass spectrometry. We observed a comparable population of α and β glycated tetramers for mono-glycated HbA0. Surprisingly, doubly and triply glycated HbA0 also showed mono-glycated α and β globins. Thus, we propose that glycation of hemoglobin (HbA) occurs symmetrically across α and β globins with preference to unmodified globin first. Correlation between conventional and mass spectrometry-based quantification of GHb showed a reliable estimation of the glycemic index of individuals carrying HbA0. Mutant HbAs have different retention time than HbA0 due to the differences in their surface charge. Thus, their glycated analog may elute at different retention time compared to GHb. Consequently, our method would be ideal for assessing the glycemic index of an individual carrying mutant HbA.