title: Detection of a thalassemic alpha-chain variant (Hemoglobin Groene Hart) by reversed-phase liquid chromatography.
abstract: BACKGROUND
  Hemoglobin (Hb) Groene Hart [alpha119 (H2)Pro-->Ser (alpha1)], also known as Hb Bernalda, is a nondeletional alpha-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 alpha-thalassemic behavior of this variant has been explained as an impaired interaction between the alpha-globin chain and the alpha-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 alpha(A)-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 -alpha(3.7) deletion). These patients possessed abnormal hematologic features suggesting an alpha-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.
authors.affiliation: Institut de Biologie et de Biochimie des Protéines, IFR128, Université Claude Bernard-Lyon I, Lyon, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Zanella-Cleon
authors.forename: Michel
authors.initials: M
authors.lastname: Becchi
authors.forename: Philippe
authors.initials: P
authors.lastname: Lacan
authors.forename: Piero C
authors.initials: PC
authors.lastname: Giordano
authors.forename: Henri
authors.initials: H
authors.lastname: Wajcman
authors.forename: Alain
authors.initials: A
authors.lastname: Francina
mesh_terms: D000328:Adult; D002675:Child, Preschool; D002853:Chromatography, Liquid; D006455:Hemoglobins, Abnormal / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013789:Thalassemia / Q000175:diagnosis*
chemical_list: D006455:Hemoglobins, Abnormal; C475066:hemoglobin Groene Hart