This is an extension of the Fellowship: 'NISA: Novel approaches for in situ analysis of biomolecules' (EP/L023490/1).
The aim of the original research was to develop novel approaches for in situ biomolecular analysis, i.e., the analysis of biomolecules directly from their natural (or actual) environment. The principal focus has been on the in situ analysis of proteins. Proteins are the work-horses of the cell and perform all the functions required for life. They also find uses as therapeutics and in consumer products. To gain insight into the roles of proteins in life processes, it is necessary to analyse proteins at a molecular level. Mass spectrometry, in which ionised molecules are characterised according to their mass-to-charge, is ideally suited to this challenge, offering high sensitivity, broad specificity (all molecules have a mass), and the capability for chemical structure elucidation.
The majority of research within the original fellowship has concentrated on development of mass spectrometry tools for in situ analysis of INTACT, but UNFOLDED, proteins. Significant advances in sensitivity have been achieved through hyphenation of mass spectrometry with gas-phase separation techniques and modifications to the mass spectrometry instrumentation. These tools enable identification of unknown proteins, identification and localisation of sites of protein modification or mutation, and spatial profiling (mass spectrometry imaging) of proteins within the substrate. Those tools do not, however, provide information on the overall 3-D structure of proteins. It is the 3-D structure of proteins that dictate their function. Knowledge of protein structure is therefore vital in deciphering the roles of protein in health and disease. In order to fully interrogate the relationship between protein structure, function and environment, it is necessary to develop tools incorporating native mass spectrometry in which proteins remain in their FOLDED form and their inter- and intra-molecular noncovalent interactions are maintained. To address that need, preliminary research undertaken as part of the original fellowship has focused on developing methods for NATIVE AMBIENT MASS SPECTROMETRY in which folded proteins, protein complexes and protein assemblies are sampled directly from their physiological environment. To date, our research in this area has focused on a single sampling technique, i.e., liquid extraction surface analysis; however, there are many ambient sampling approaches which may prove suitable, each offering different specifications in terms of sensitivity, speed, and spatial resolution.
The aim of the fellowship extension is to establish NATIVE AMBIENT MASS SPECTROMETRY as a broad discipline for the in situ analysis of folded proteins and their complexes. The goal is to develop a suite of tools which will be capable of providing information on protein function in health and disease. Each potential application of native ambient mass spectrometry will come with its own unique challenges. For example, spatial resolution i.e., intricate mapping of the protein distribution in the tissue, may be the crucial requirement. Alternatively, high throughput (speed of analysis) may be the key to success, or it may be that the sensitivity of the technique that is vital. By widening the scope of native ambient mass spectrometry to encompass a full range of sampling techniques, we will enable each of these challenges to be addressed. Moreover, a range of ion mobility spectrometry techniques, which enable measurement of protein structure as well as improving sensitivity, will be integrated with native ambient mass spectrometry allowing spatial profiling of 3D protein structure. The impact of the research will be demonstrated by application to Alzheimer's disease, a disease associated with protein misfolding and aggregation, and non-alcoholic fatty liver disease, a disease associated with unusual binding between proteins and lipids.
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