New high resolution mass spectrometry facilities for macromolecules and metabolites at the University of East Anglia

Lead Research Organisation: University of East Anglia
Department Name: Chemistry

Abstract

Mass spectrometry has revolutionized many aspects of research in the molecular sciences, through its ability to unambiguously identify molecules by measuring their mass with incredible accuracy. UEA researchers have pioneered the use of a particular type of mass spectrometry in studies of proteins, fascinating workhorse molecules of life that are dynamic in the way they interact with each other and with small molecules. Many proteins contain fragile metal cofactors that are able to react chemically in ways that proteins alone cannot. These cofactors are often pre-assembled (by complex pathways) and then inserted into the protein much like a factory assembly line. The chemistry that goes on at these cofactors often involves changes in mass that controls protein function. UEA researchers have used a type of gentle mass spectrometry that preserves the fragile cofactors, and have been able to 'watch' reactions happen by measuring the resulting usually small changes in mass, providing unprecedented insight into processes that are essentially invisible to other methods. This work has revealed how bacteria sense key environmental ques such as oxygen and iron availability, which are known to be virulence factors that regulate infection. The aim of this bid for a new mass spectrometer with state of the art capabilities is to apply mass spectrometry to a broader range of cofactor-containing proteins that cannot be studied using existing limited facilities. Proposed projects include understanding the assembly of the hydrogenase cofactor and the development of biohybrid materials, both of which are relevant to bioenergy, small molecules that bind DNA as tools and potential therapeutics, and the discovery of novel natural antifungal molecules that may have potential for use in the protection of valuable crops. The new instrument will benefit researchers at UEA more broadly as it will make accessible technologies that are not currently available.

Technical Summary

Mass spectrometry (MS) has revolutionized many aspects of the molecular sciences, through its ability to provide accurate mass information, permitting unambiguous identification of molecules. Using regular MS solvents macromolecules such as proteins lose their native structures and any non-covalent interactions (eg with cofactors, proteins or DNA). The application of MS under non-denaturing ('native') conditions allows macromolecules to maintain their folded conformation, such that non-covalent interactions are maintained. We have pioneered native MS studies of protein-metallocofactor reactivity, resulting in unprecedented insight into function and illustrating the power of native MS for broader application in studies of larger metalloprotein complexes, interactions between proteins and proteins and small molecules, and those involving DNA. For small molecule work and metabolite discovery, a hybrid MS/MS configuration is needed, which enables ions to be selectively fragmented, thus aiding unambiguous identification. A new MS facility providing state of the art sensitivity and resolution, particularly at higher m/z values, will enable our pioneering native MS work to continue and will provide for a range of other projects at UEA, for which high resolution MS is essential. The research programme focusses on three themes: Metallo-cofactor biochemistry underpinning bioenergy and climate change (Theme 1); DNA structures and interactions for tools and therapeutics (Theme 2); and, Metabolomics approaches (Theme 3), in which natural metabolites, eg with anit-fungal properties, will be identified. The availability of new MS facilities will open up major research avenues not currently available or practical at UEA.

Planned Impact

This application for a state of the art high resolution mass spectrometer (HRMS) instrument will support a range of projects at the University of East Anglia that relate to themes in Metallo-cofactor biochemistry underpinning bioenergy and climate change (Theme 1), DNA structures and interactions for tools and therapeutics (Theme 2), and Metabolomics approaches (Theme 3). These projects will have diverse and far reaching impacts within the UK and internationally.
Outside of academia, there are several groups of potential beneficiaries, including:
- policy makers and commercial stakeholders, who are likely to be interested in the anticipated advances made in the described projects. These include advances in understanding fundamental aspects of how the metal cofactors of key metalloenzymes are assembled. The focus here is on hydrogenase, which is centrally important to efforts to develop a hydrogen economy, and nitrous oxide reductase, which is the only enzyme know capable of consuming the major greenhouse gas nitrous oxide (levels of which have risen sharply due to intensive farming methods). These, together with anticipated advances in the development of biohybrid materials for capture of solar energy and its transfer/storage will impact on energy and climate change policy, and associated commercial developments. Advances in the development of small molecules that bind DNA with high specificity will be of interest to the pharmaceutical industry because this could open up novel therapeutic strategies. Advances in the development of novel anti-fungals that protect commercially important wheat crops could, in the longer term, lead to development of strategies in which seeds are coated in anti-fungal producing bacteria that forms a symbiotic relationship with the plant host. This would be of huge interest to the agro-tech sector. Finally, the anticipated advances towards characterising the adductome, and the effect of nutrition and diet on it, although a long-term project, would be of major interest to the healthcare and food industries.
- the biotechnology and pharmaceutical sectors and public sector laboratories, from the point of view of benefiting from future employment of researchers (PDRAs, PhD students, and undergraduates working within the research groups) trained in state-of-the-art mass spectrometry methods, particularly novel native HRMS methods;
- schools and the general public, who benefit from engagement activities running parallel with the research effort, which seek to inspire the next generation of science undergraduates and scientists and to better inform the general public of key scientific concepts and issues over which society has an influence. For example, the vital roles that metal ions play in maintaining health, in developing next generation energy materials, and in mitigating the effects of climate change are not well appreciated by the general public. Successful science festival exhibitions focused on the discovery of novel antibiotics from Streptomyces living in natural habitats illustrates the impact these activities can have.

Publications

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