Abstract

Sickle cell disease is a genetic disorder caused by a point mutation in the β-globin gene on chromosome 11 where thymine replaces adenine resulting in the amino acid substitution of valine for glutamic acid in residue 6 (difference between HbA and HbS). Despite the occurrence of the single known point mutation in all the HbSS patients, there is tremendous heterogeneity in the clinical severity and outcome of the disease. In order to investigate the cause of this diversity, it would be extremely useful to compare alterations in the Red Blood Cell (RBC) proteome versus variation in vaso-occlusive crisis rate or Acute Chest Syndrome (ACS) rate by protein profiling techniques. The three protein profiling techniques being performed by the Goodman laboratory on the RBC membrane proteome are: 2D DIGE (two dimensional differential gel electrophoresis), cICAT (cleavable isotope coded affinity tags) and iTRAQ (Isobaric tags for relative and absolute quantification). The iTRAQ technique is a protein profiling method that can analyze four or eight samples simultaneously thus minimizing sample to sample variation. Before addressing sickle cell severity induced changes in the RBC proteome, our primary step is to measure the variation in the iTRAQ methodology, including any protein to protein and peptide to peptide variation. This is the focus of my Masters Thesis.