The use of mass spectrometry and DNA technology in the investigation of hemoglobin disorders

Abstract: Hemoglobin (Hb) disorders, which severely affect nearly 300,000 newborns every year, have become a global problem. The disorders manifest a wide spectrum of clinical symptoms ranging from asymptomatic to lethal conditions. Inheritance or spontaneous occurrence of genetic defects could lead to either a structural abnormality of Hb (Hb variants) or an impaired or no synthesis of the globin chains (thalassemias). The majority of Hb variants are caused by a single nucleotide substitution resulting in an amino acid change in the globin chain, while the thalassemias are caused by a diverse array of mutations. The study of Hb disorders can elucidate the structure- function relationship of the Hb and may clarify the mechanism of pathogenesis. Hb disorders are good models of protein abnormalities that can be directly or indirectly associated with molecular diseases. Conventional electrophoresis and HPLC techniques give presumptive identification of Hb variants and are still widely used today. Modem developments in DNA technology have revolutionized detection and identification of Hb disorders at the nucleotide level. Simultaneously, mass spectrometry has emerged as a powerful tool in the field of biological and biomedical studies. Proteins of almost unlimited molecular mass can be analyzed and amino acid sequence of peptides can be readily determined. In order to evaluate the performance and limits of DNA and electrospray mass spectrometry techniques in the investigation of Hb variants, different studies using these techniques were initiated. The mass spectrometric experiments were done at two levels, i.e. at the intact globin chain and at the tryptic peptide levels. The mass analyses were performed on twostep diluted whole blood samples requiring only 10 µL of the whole blood. Blood samples stored at +4° C or -20° C were used. The variants which were present to greater than 10% of the total Hb, and whose masses differed by greater than 6 Da from the normal chain were in most cases readily identified by electrospray mass spectrometry. Data from isoelectric focusing and a knowledge of the genetic code greatly aided the characterization of Hb variants. Accurate mass measurements (<±5 ppm) of intact globin chains (~16 kDa) allowed for detection of the variant chains that differ in mass by <6 Da from the corresponding normal globin chain. A good correlation was found between the HPLC and mass spectrometric methods in quantifying Hb variants. Tandem mass spectrometry (MS/MS) of intact beta-chains identified some Hb variants due to an amino acid change within 40 to 50 residues from the terminals of the globin chain. In others, the MS/MS data directed the location of the amino acid substitution to a certain region of the beta- globin chain. A secondary structural change in Hb variants, i.e. disulfide bridge, was also demonstrated and characterized by mass spectrometry. However, due to the complexity of the peptide mixture, DNA analyses were sometimes required to allow successful interpretation of mass spectrometry data. In general, mass spectrometry was a powerful tool for screening and identification of aberrant Hb proteins.