Analysis of red cell membrane proteins by capillary gel electrophoresis

Background. Several hereditary hemolytic anemias associated with the abnormal red cell shapes, such as hereditary spherocytosis (HS) and elliptocytosis (HE) are caused by defects of red cell membrane proteins. The detection of these abnormalities is usually performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (1), a time-consuming and lab-intensive technique. Therefore, aim of this study was to set up a method based on SDS-capillary gel electrophoresis (SDS-CGE) for the separation and quantification of major erythrocyte membrane proteins. Methods. Twenty whole blood samples in EDTA without any apparent sign of hemolysis were collected from the laboratory routine and processed within 2 days. Erythrocytes were separated from platelets and leukocytes and then subjected to a hypotonic treatment (2) in order to prepare red cell ghosts. The analyses of red cell membrane proteins were carried out with a Beckman Coulter ProteomeLab PA800 capillary electrophoresis apparatus equipped with a diode array detector set at 220 nm. The SDS-MW Analysis Kit (Beckman Coulter Inc.), including molecular mass standard proteins, was used. The abundances of the main membrane proteins were quantified by expressing their relative concentrations respect to band 5 (actin). Results. Seven major erythrocyte membrane proteins were separated and identified: α- and β-spectrin, and bands 3, 4.1, 4.2, 5 and 6. The molecular weights of all these proteins were in good agreement with those reported in literature (3). Reproducibility (expressed as CV, %) of the migration times were between 0.1 % and 0.2 %. Reproducibility of the relative abundances of the main red cell membrane proteins were between 4.5 % (band 4.1/band 5) and 16.4 % (band 6/band 5). Conclusions. The preliminary results obtained in our investigation prove that SDS-CGE may be an alternative approach to standard SDS-PAGE for the identification of red cell membrane disorders.