Alterations of the erythrocyte membrane proteome and cytoskeleton network during storage - a possible tool to identify autologous blood transfusion

Mature red blood cells (RBCs) are the end-stage of a development process that starts in the bone marrow and continues to differentiate, through reticulocyte stage, entering into the circulation with a four-month lifespan. While stored, RBCs undergo different changes. The aim of this study was to evaluate changes occurring in RBC membranes during storage that could be used as possible markers to detect the misuse of blood transfusion in sports. Whole blood was collected from two volunteers in blood bags and stored for 42 days at 4 degrees C. At different times (1, 7, 21, and 42 days of storage) whole blood was extracted under sterile conditions and submitted to RBC membrane ghost preparation and further analysis. Proteomic methods were applied using two strategies: protein oriented using 2-DE gels and peptide oriented using isobaric tags for relative and absolute quantitation (iTRAQ). In both approaches, the goal was to compare detectable changes in RBC membrane proteome before and after standard storage at different times. Some of the changes were confirmed with both methodologies employed, while with others only with one of them. Complementarities of the methods in this case showed to be an advantage. Changes were observed in two different protein complexes. In one of them, changes consisted of proteins decreasing, while increasing in the other during storage of RBCs. They are mostly located in cytoskeleton - spectrin beta, band 4.2, ankyrin-1, tropomodulin-1, beta adducin, band 4.9 (dematin), tropomyosin, while some changes were also observed in transmembrane proteins (glycophorin C, aquaporin-1, band 3). Copyright (C) 2012 John Wiley & Sons, Ltd.