Characterization of the elusive disulfide bridge forming human Hb variant: Hb Ta-Li beta83 (EF7)Gly --> Cys by electrospray mass spectrometry.
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 (beta83Gly --> Cys), Cys83 forms inter-molecular disulfide bridges. Routine analysis of the denatured Hb showed the presence of a minor beta 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 beta globins. The mutation was localized to peptide betaT10 by analysis of a tryptic digest. Tandem mass spectrometry and DNA sequencing confirmed the Gly --> Cys substitution occurred at residue 83 of the beta chain. Problems encountered in identifying the components in mixtures of monomers and dimers are discussed.
Rai
Dilip K
DK
Department of Medical Laboratory Sciences and Technology, Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden.
Landin
Britta
B
Griffiths
William J
WJ
Alvelius
Gunvor
G
Green
Brian N
BN
D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D005914:Globins / Q000032:analysis; D006455:Hemoglobins, Abnormal / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007525:Isoelectric Focusing; D008745:Methylation; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D021241:Spectrometry, Mass, Electrospray Ionization; D013439:Sulfhydryl Reagents; D014357:Trypsin
D004220:Disulfides; D006455:Hemoglobins, Abnormal; D013439:Sulfhydryl Reagents; C451248:hemoglobin Ta-Li beta83 (EF7)Gly-Cys; D005914:Globins; D014357:Trypsin; D004229:Dithiothreitol