AbstractTo improve detection of the misuse of erythropoietin (EPO) for performance enhancing purposes, this PhD examined ways to improve recovery and preanalysis concentration of EPO from urine. It also looked at ways to enhance the signal in liquid chromatography tandem mass spectrometry of the acidic glycopeptides from digested EPO, and at distinguishing between recombinant EPO and human urinary EPO based on differences in their glycosylation. Due to a shortage of supply of available analytical standards, model glycoproteins were frequently used in place of endogenous EPO.
Immunoextraction with magnetic beads effectively recovered EPO from urine which had been filtered to remove large proteins, but was unsuccessful from unfiltered urine, suggesting more research into the right choice of antibody was needed.
The specific and reversible binding of boronic acids to cis-diol groups found in the glycan groups of glycoproteins was investigated as a device for the selective binding of EPO. Attempts were made to functionalise mesoporous silica for use as a column packing material. Although there was evidence that at least one method of functionalisation was successful, the use of this silica to extract glycoproteins and glycopeptides was not.
Signal enhancement through the introduction of ‘superchargers’ into LC solvents was investigated. This was effective with small molecules, and also improved detection of sialylated glycopeptides. The results do not fit entirely with current models of how superchargers exert their effect, suggesting they are incomplete.
Finally, the cleavage, digestion and derivatisation of N-glycans to identify bisected and non-bisected structures as a way to discriminate between rEPO and huEPO was examined. Samples were analysed using LC-MS and CE-LIF, and although much of the work was carried out using a model glycoprotein, there is some evidence that the approach may be capable of discriminating between artificial and endogenous EPO at the levels found in anti-doping samples.
|Date of Award
|David Cowan (Supervisor) & Mark Parkin (Supervisor)