Structural analysis of glycated human hemoglobin using native mass spectrometry

Authors : Muralidharan M, Bhat V, Mandal AK

Publication Year : 2019

Abstract :

Glycated hemoglobin (HbA1c ), is the indicator of the long-term glycemic index of an individual. HbA1c is formed by the irreversible modification of N-terminal à-amino group of ß globin chain with glucose via Amadori rearrangement. Cation exchange chromatography exploits the difference in surface charges between HbA1c and native hemoglobin (HbA0 ) for their separation and quantification. However, glucose condensation is specific to primary amino groups. Therefore, structural characterization of HbA1c synthesized in-vivo is essential as multiple glycation may interfere with HbA1c assessment. The stoichiometric composition of different glycated hemoglobins from a 19% HbA1c sample was deduced using native mass spectrometry. We observed a comparable population of à and ß glycated tetramers for mono-glycated HbA0 . Surprisingly, doubly- and triply-glycated HbA0 also showed mono-glycated à and ß globins. Thus, we propose that glycation of hemoglobin occurs symmetrically across à and ß globins with preference to unmodified globin first. Correlation between conventional and mass spectrometry-based quantification of HbA1c showed a reliable estimation of the glycemic index of individuals carrying HbA0 . Mutant hemoglobins 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 HbA1c . Consequently, our method would be ideal for assessing the glycemic index of an individual carrying mutant hemoglobin.