Characterization of the elusive disulfide bridge forming human Hb variant:Hb Ta-Li β83 (EF7)Gly → Cysby electrospray mass spectrometry

Abstract

An electrospray mass spectrometric approach to the identification of a human hemoglobin (Hb) variant involving a Cys residue incorporation is presented. In Hb Ta-Li (β83Gly → Cys), Cys83 forms inter-molecular disulfide bridges. Routine analysis of the denatured Hb showed the presence of a minor β chain variant whose mass apparently was 1 Da less than the expected mass difference of 46 Da for a Gly → Cys substitution. Reduction of the globin chains with dithiothreitol gave an intense monomer with the expected mass difference for the Gly → Cys substitution. After reprocessing the original raw data from the denatured Hb and taking into account the possibility of dimer formation, a component was revealed whose mass was consistent with a disulfide linked dimer of Ta-Li β globins. The mutation was localized to peptide βT10 by analysis of a tryptic digest. Tandem mass spectrometry and DNA sequencing confirmed the Gly → Cys substitution occurred at residue 83 of the β chain. Problems encountered in identifying the components in mixtures of monomers and dimers are discussed.