Surface plasmon resonance on-line with liquid chromatography – mass spectrometry for the quantification and identification of proteins

Abstract

Although SPR-MS as a system for studying molecular interactions, e.g. ligand fishing, orphan receptor and drug screening, has been in use since 1997, several aspects remain to be improved. Among these are non-specific adsorption (NSA) and surface modification allowing protein coupling. These are related, as they concern sensor-surface coating allowing both the immobilisation of receptor molecules as well as preventing unwanted interactions. In the case of an on-line SPR-LC-MS/MS system, an interface between the SPR and the LC system is required. Additionally, there is need for a means of efficient digestion of isolated proteins in order to allow tandem-MS identification. The topics that were studied in this thesis arose from these subjects. First surface-coating development was performed for both sensors and enzyme reactors. The developed sensors and reactors were characterised. Next, the integration of all necessary instruments was investigated, ultimately evolving into a proof of principle. Lastly, an application was developed in which the isolation, quantification and identity confirmation of β2-microglobulin in urine was performed.