oa Development Of A Novel Method For The Selective Detection Of Erythrocyte Membrane Bound Proteins

Blood doping still remains as a performance enhancement strategy misused by athletes . Blood doping is the practice of boosting the number of red blood cells in the bloodstream by means of either blood transfusion of an individual or by administration of erythropoietin, in order to increase the oxygen carrying capacity of the blood. There is no direct method currently for the detection of autologous transfusion, leaving this an open issue in antidoping strategies. Recently it was reported that some proteins might represent suitable markers for in vitro aging of erythrocytes, particularly those proteins that can be detected in the membrane. Thus, this study was conducted on erythrocytes isolated from fresh and 20-day old blood samples of the same individual, with a particular focus on the erythrocyte membrane bound proteins. Isolated proteins were separated into 12 fractions on the Gelfree system, digested with trypsin and separated using a nanoLC. Eluted peptides were then spotted on a MALDI plate with MALDI spotter and detected on MALDI-TOF/TOF instrument. Afterwards, the MS/MS data for these peptides were identified using SwissProt and Mascot database search. The results have shown significant differences in the peptides when comparing fresh and stored blood samples, revealing several potential targets that can be used as markers for blood doping detection. The next step is to prove the reproducibility of the results on a larger number of specimens as an initial validation step.