Electrospray ionization-tandem mass spectrometry analysis of peptides derived by enzymatic digestion of oxidized globin subunits: An improved method to determine amino acid substitution in the hemoglobin “core”

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The determination of an amino acid substitution in the "core" region of abnormal hemoglobin is technically difficult and is time- and labor-intensive both by conventional and by mass spectrometry techniques. Underivatized subunits cleaved with trypsin and lysyl endopeptidase were analyzed by high-performance liquid chromatography-electrospray ionizationmass spectrometry. The core peptides showed a series of multiply charged ions of homo- and heterodimers. Abnormal peptides in the core region could be detected in dimeric form. The sequence of core peptides was determined by product ion spectra of the peptides from oxidized globin digested with trypsin and lysyl endopeptidase. Oxidation of cysteine residues to cysteic acids in the core region resulted in the strong promotion of y-series ions by product ion analysis with a tryptic peptide from apoprotein B-100 as previously reported by Burlet, Yang, and Gaskell (J. Am. Soc. Mass Spectrom. 1992, 3, 337-344). These techniques were used to prove that substitution of an unstable hemoglobin, known as Hb Santa Ana (β88 leucine → proline), occurred in a patient with congenital hemolytic anemia. The tandem mass spectrometry analysis with oxidized globin digested with trypsin and lysyl endopeptidase offers a novel method to detect substitutions in the core region of hemoglobin.