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Detection of a Thalassemic α-Chain Variant (Hemoglobin Groene Hart) by Reversed-Phase Liquid Chromatography
- Source :
- Clinical Chemistry. 54:1053-1059
- Publication Year :
- 2008
- Publisher :
- Oxford University Press (OUP), 2008.
-
Abstract
- Background: Hemoglobin (Hb) Groene Hart [α119 (H2)Pro→Ser (α1)], also known as Hb Bernalda, is a nondeletional α-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The α-thalassemic behavior of this variant has been explained as an impaired interaction between the α-globin chain and the α-Hb–stabilizing protein.Methods: To separate globin chains, we developed a modified reversed-phase liquid chromatography (RPLC) procedure that uses acetonitrile–water solvents containing up to 3 mL/L trifluoroacetic acid. After RPLC, we characterized the isolated globin chains by electrospray ionization (ESI) mass spectrometry (MS) and analyzed their tryptic peptides with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nano-LC–ESI–MS/MS.Results: RPLC detected an abnormal peak with a retention time substantially greater than that of the wild-type αA-globin chain. We identified this variant as Hb Groene Hart and found it in the hemolysates of 11 unrelated patients (1 homozygote, 9 heterozygotes, and 1 heterozygote associated with the −α3.7 deletion). These patients possessed abnormal hematologic features suggesting an α-thalassemia phenotype. Molecular modeling suggested that the increase in hydrophobicity was due to opening of the GH interhelical segment following replacement of amino acid residue 119 with a nonhelix breaker residue.Conclusions: This method allows the detection of Hb variants at low concentrations, and adjusting the composition of the organic solvents enables the method to identify Hb variants with large changes in hydrophobicity.
- Subjects :
- Adult
Models, Molecular
Spectrometry, Mass, Electrospray Ionization
Hemoglobins, Abnormal
Electrospray ionization
Clinical Biochemistry
Tandem mass spectrometry
chemistry.chemical_compound
Tandem Mass Spectrometry
Trifluoroacetic acid
medicine
Humans
Globin
Chromatography
Chemistry
Biochemistry (medical)
Reversed-phase chromatography
medicine.disease
Matrix-assisted laser desorption/ionization
Hemoglobinopathy
Child, Preschool
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Mutation
Thalassemia
Hemoglobin
Chromatography, Liquid
Subjects
Details
- ISSN :
- 15308561 and 00099147
- Volume :
- 54
- Database :
- OpenAIRE
- Journal :
- Clinical Chemistry
- Accession number :
- edsair.doi.dedup.....cea89ea8424637fb1eab919e26b599b6
- Full Text :
- https://doi.org/10.1373/clinchem.2007.097857