Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within αT9 peptide : Identification of Hb Villeurbanne α89 (FG1) His → Tyr

Mutation-induced amino acid exchanges occurring on the large T9 peptide of the α-chain of human hemoglobin (residues 62–90) are difficult to identify. Despite their highm/zvalue (aroundm/z3000), collision-induced dissociation spectra of liquid secondary ion mass spectrometrically generated protonated αT9 peptides were performed successfully. In parallel electrospray mass spectrometry (MS) was used both to measure the molecular mass of the intact proteins and to determine the number of protonatable sites in the αT9 peptides. Peptide ladder sequencing using carboxypeptidase digestions and analysis of the truncated peptides by matrix-assisted laser desorption ionization time-of-flight MS confirmed the interpretation. This set of methods allowed the characterization of three hemoglobin variants, with amino acid exchanges located in the αT9 part of the sequence. Two of them, Hb Aztec [α76(EF5) MetThr] and Hb M-Iwate [α87(F8) HisTyr] were already known. The third [α89(FG1) HisTyr] was novel and named Hb Villeurbanne. © 1997 by John Wiley & Sons, Ltd.