MSACL Conference Schedule

Identifying Proteolytic Peptides in Thalassemic Red Blood Cells
Wed 12:00 PM - Session: Disease Markers
Kai Tang
Laboratory investigations of hemoglobinopathies usually begin with initial routine screening such as complete blood count (CBC) and hemoglobin (Hb) quantification. Preliminary results obtained serve as a guide for more specific diagnostic procedures. In cases of alpha-thalassemias, it is often difficult to identify disease genotypes until verification by DNA-based techniques. For stop-codon mutations like Hb Constant Spring (CS) and Hb Pakse (PS), the abnormal elongated globin can be detected in homozygotes and compound heterozygotes with alpha-thalassemia 1 by using high performance liquid chromatography (HPLC). However, in heterozygotes, compound heterozygotes with alpha-thalassemia 2 and co-inheritance of beta-thalassemia, it is usually not possible to detect the HCS or HPS allele by HPLC due to its limited sensitivity. We chose to search for proteolytic peptides in red blood cells by mass spectrometry. We also tried to detect the intact elongated alpha-hemoglobin protein directly by mass spectrometry in an attempt to differentiate Hb CS and PS. The signature peptide approach identified proteolytic peptides from these unstable hemoglobins, which corresponded to the C-terminal fragments of the elongated alpha globin, with intense peaks appearing at 1638.7 and 2010.1 Da. They have been identified in all samples carrying the Hb CS or PS allele, including heterozygotes and CS double heterozygotes with HbE. With their dominance and consistency, these signature peptides have the potential to serve as biomarkers for alpha-thalassemic patients with stop-codon mutations. Direct molecular weight measurement of the intact alpha-globin chain provides complementary information about the stop codon mutation types for homozygous and Hb H-CS or Hb H-PS disease samples.

(Research supported by BMRC grant from A*STAR, Singapore)