COMBINED MASS-SPECTROMETRIC METHODS FOR THE CHARACTERIZATION OF HUMANHEMOGLOBIN-VARIANTS LOCALIZED WITHIN ALPHA-T9 PEPTIDE - IDENTIFICATION OF HB VILLEURBANNE ALPHA-89 (FG1) HIS-]TYR

Mutation-induced amino acid exchanges occurring on the large T9 peptid e of the alpha-chain of human hemoglobin (residues 62-90) are difficul t to identify. Despite their high m/z value (around m/z 3000), collisi on-induced dissociation spectra of liquid secondary ion mass spectrome trically generated protonated alpha T9 peptides were performed success fully. In parallel electrospray mass spectrometry CMS) was used both t o measure the molecular mass of the intact proteins aml to determine t he number of protonatable sites in the alpha T9 peptides. Peptide ladd er sequencing using carboxypeptidase digestions and analysis of the tr uncated peptides by matrix-assisted laser desorption ionization time-o f-flight MS confirmed the interpretation. This set of method allowed t he characterization of three hemoglobin variants, with amino acid exch anges located in the alpha T9 part of the sequence. Two of them, Hb Az tec [alpha 76(EF5) Met --> Thr] and Hb M-Iwate [alpha 87(F8) His --> T yr] were already known. The third [alpha 89(FG1)His --> Tyr] was novel and named Hb Villeurbanne. (C) 1997 by John Wiley & Sons, Ltd.