Unique tryptic peptides specific for bovine and human hemoglobin in the detection and confirmation of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers (HBOCs) of bovine hemoglobin (Hb) or human Hb origin were developed for replacement or augmentation of blood during transfusion and have the potential to increase oxygen-carrying capacity of circulating blood and thus improve tissue oxygen delivery. Due to their potential for increasing oxygen-carrying capacity of circulating blood, they are excellent candidates for abuse in human and equine athletes. To deter athletes from blood doping with HBOCs such as Hemopure and Oxyglobin (OXY), a method for detection, confirmation, quantification, and distinguishing of HBOCs from native hemoglobin in test samples is needed. The purpose of this study was to identify unique peptides specific for bovine Hb and human Hb that are useful in the detection and confirmation of HBOCs in test samples. The LC-MS chromatographic peak profiles of tryptic digests from OXY, bovine Hb, human Hb, and equine Hb were compared, and unique tryptic peptides specific for bovine Hb, human Hb, and equine Hb were identified. The peptides specific for bovine Hb and OXY are related to bovine Hb alpha chain residues 69-90 and beta chain residues 40-58. The peptides specific for human Hb are related to human Hb alpha chain residues 63-91 and beta chain residues 42-60 and 68-83. The amino acid sequences of these unique tryptic peptides were confirmed by their characteristic MS/MS spectra. MS/MS spectra, b-ion series and y-ion series, and LC retention time of the tryptic peptides are essential pieces of information for the unequivocal identification, detection, and confirmation of HBOCs. The results of this study provide useful and defensible data on identification, detection, and confirmation of HBOCs of bovine Hb or human Hb origin. In addition, in-ESI-source fragmentation of tryptic peptides was observed in this study. The fragmentation was undesired since it decreased intensities of the trypic peptide ions, but it was helpful to elucidating sequences of the tryptic peptides thanks to the fragment peptide ions produced from the fragmentation.