Tandem Mass Tag mass spectrometry analysis of protein levels in band 3 null RBC membranes

Abstract

The band 3 null COIMBRA patient has been studied previously in order to understand the main causes of this condition. In this study, ghost membrane and cytoskeleton proteins were isolated from red blood cell (RBCs) of the band 3 null individual and three controls were compared. Using Tandem Mass Tag (TMT) mass spectrometry, in order to identify proteins that were increased or decreased in their expression in the band 3 null RBC membranes relative to controls. As expected, band 3 expression was low in the band 3 null cells relative to the controls, as were proteins of the band 3 macrocomplex. This study confirmed the previously reported reduction in ankyrin-1, RhAG, CD47, and band 4.2. However, GPA, Rh polypeptide and ICAM4 varied compared to controls and α- and β-spectrin were not found to be reduced. This discrepancy may reflect differences in methodology and/or Rh phenotype of the controls. This study found a number of novel proteins that were decreased in band 3 null RBC membranes. These included two cell cycle associated proteins, AGO2 and ATM, and membrane proteins TTN, SNX32, and CYBR1. These proteins have been reported to be involved in cytokinesis, vesicle trafficking, ascorbate recycling and RBC structure. Furthermore, numerous novel proteins were found to be increased in band 3 null RBC membranes relative to controls including NMIIA, FLNA, and TUBA1C, which may contribute to enucleation and/or reticulocyte maturation. Also increased were TMCC2 and SLC1A5, SLC7A5 and PSAP, and HSP70, proteins associated with erythropoiesis, lysosomes and cell stress respectively.