N-Terminal Adducts of Bovine Hemoglobin with Glutaraldehyde in a Hemoglobin-Based Oxygen Carrier

Abstract

Hemoglobin-based oxygen carriers (HBOCs) are being developed for the medical field, but because they could increase an athlete's performance, they are misapplied for doping purposes. We previously presented a screening method to detect Oxyglobin (Biopure Corp.) and PolyHeme (Northfield Laboratories Inc.) in serum samples using total acid hydrolysis followed by electrospray mass spectrometry analyses. An alternative mass spectrometric method involving enzymatic hydrolysis is here presented. Digestion of Oxyglobin by endoproteinase Glu-C and LC/MS analyses of the mixture allowed the detection of unique peptidic fragments in comparison with a bovine hemoglobin digest. Tandem mass spectrometry experiments of these peptide ions were performed, and two specific species were actually identified as the N-terminal enzymatic fragment of the beta chain carrying two different modifications. Sequential MS3 experiments using an ion trap mass spectrometer permitted us to locate the chemical modification by the glutaraldehyde on the NH2-terminal group and to propose a structure for the modified peptides. In another set of experiments, screening of these two diagnostic ions into Oxyglobin-spiked serums using precursor ion scan mode in a triple quadrupole instrument allowed the detection of this HBOC with a detection limit of 2 g L(-1).

Mass spectrometry in sports drug testing: Structure characterization and analytical assays

“…Additionally, the digestion of plasma samples with endoproteinase Glu-C followed by LC-MS/MS analysis with precursor ion scanning was used to identify Nterminal modifications of bovine Hb due to the use of crosslinking reagents. The peptide MLTAEE generated by enzymatic hydrolysis bears a modified amino function of the N-terminal methionine, which gives rise to precursor ions at m/z 759 and 761 to generate characteristic product ions at m/z 170 and 172, respectively, which are employed as target product and precursor ions in doping control assays (Gasthuys, Alves, & Tabet, 2005).…”

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing

“…While distinct guidelines and so‐called identification points (IPs) have been defined for low molecular weight substances, LC/MS/MS is now being more widely used for peptide hormone and protein identification,6–13 necessitating rules for this class of compounds. The task of establishing identification criteria has been assigned to the accredited drug testing laboratories; hence, we discuss a set of criteria for target peptide and protein analysis from blood and urine in doping control specimens.…”

Identification and Characterization of Peptides and Proteins in Doping Control Analysis

“…Although scientific data about the effect of HBOCs on endurance and athletic performance is very limited and skepticism regarding a positive influence is prevalent (Schumacher et al, 2001;Schumacher and Ashenden, 2004), artificial oxygen carriers are considered relevant for doping controls. Numerous studies have been published demonstrating the determination of HBOCs in human plasma by means of various techniques such as 1D-gel electrophoresis , size-exclusion chromatography combined with UV detection (Gotzmann et al, 2002;VarletMarie et al, 2004) as well as different mass spectrometric approaches (Thevis et al, 2003;Gasthuys et al, 2004Gasthuys et al, , 2005Goebel et al, 2005 a,b). HBOCs are detectable in plasma specimens only; thus, all assays are based on blood sampling Fig.…”

“…Tabet and co-workers (Gasthuys et al, 2004(Gasthuys et al, , 2005 suggested two different approaches to cope with bovine HBOCs such as Oxyglobin. The first has been based on acidic hydrolysis of serum samples followed by LC-MS/MS employing neutral loss scanning of 159 Da utilizing a triple quadrupole mass spectrometer.…”