Novel mass spectrometry-based approaches for the characterisation of systems of biological interest

Abstract

Originally established as an analytical technique in the fields of physics and chemistry, mass spectrometry has now also become an essential tool in biology. Advances in ionisation methods and novel types of instrumentation have led to the development of mass spectrometry for the analysis of a wide variety of biological samples. The work presented here describes the use mass spectrometry for the study of a number of biological systems. A new family of techniques has been developed allowing ions to be created under ambient conditions. Three of these ambient ionisation techniques, coupled to different mass analysers, were employed for the rapid screening of pharmaceutical formulations. Active ingredients were identified and subjected to collisionally induced dissociation, enabling the elucidation of potential fragmentation pathways. Drug metabolites were also successfully identified from biological samples. Inorganic mass spectrometry was employed to probe the metal centres of the enzyme, particulate methane monooxygenase, a methane-oxidising complex found in certain bacteria. This protein has been extensively studied, but questions remain regarding its catalytic mechanism, particularly the involvement of indigenous metal ions. Inductively-coupled plasma mass spectrometry experiments have indicated the presence of copper and iron within the enzyme. Protein cross-sections, obtained using ion mobility mass spectrometry, can be used to probe the conformation of molecules in the gas phase. A commercial instrument was used to investigate human hemoglobin from clinical samples. A complex assembly mechanism was deduced, resolving previous disputes in the literature, and conformational differences were observed between healthy and sickle molecules. The field of proteomics is rapidly evolving; as described, techniques are constantly being developed and improved to deal with the enormous complexity that proteomes present. Three proteomics approaches were used to study a recently identified bacterium under two growth conditions. Differences in protein expression were observed and correlated to relevant biological pathways