Simple Identification of A Cross-Linked Hemoglobin by Tandem Mass Spectrometry in Human Serum

Abstract

Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).

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

“…Mass spectrometry of hydrolyzed HBOCs employing neutral loss scanning of 159 Da utilizing a triple quadrupole mass spectrometer yielded ions at m/z 299 and 399 that were not further characterized but seemed to be generated from cross-linked bovine Hb only (Gasthuys et al, 2004). 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.…”

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.…”