Reverse Phase High-Performance Liquid Chromatography and Secondary Ion Mass Spectrometry. A Strategy for Identification of Ten Human Hemoglobin Variants

Abstract

Ten abnormal hemoglobins were detected and characterized in individual cases referred to our laboratory for evaluation of hematological problems. Six of these variants were electrophoretically silent and could be detected by reverse phase high-performance liquid chromatography (HPLC) analysis. HPLC was also used to analyze the tryptic peptides of each individual variant. In most of these variants, secondary ion mass spectra of the mixture of the tryptic peptides could reveal the aberrant peptide and predict possible substitution through the mass difference between the normal and abnormal peptide. The mass spectra of the isolated abnormal peptide generally contained sufficient fragment ions to define the position of the amino acid substitution, obviating the need for lengthy sequencing procedures. Combination of the two techniques provides a powerful tool for structural characterization of proteins. Additional information about the clinical manifestation and hematological finding in each individual case and HPLC behavior of the globin chains and their tryptic peptides are provided.