NISA: Novel approaches for in situ analysis of biomolecules

The aim of the research is to develop novel approaches for the analysis of biomolecules, and in particular proteins, directly from their natural (or actual) environment, i.e., to develop approaches for in situ biomolecular analysis. 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 various and specific roles of proteins in life processes, or to determine the therapeutic efficacy of protein drugs, or to establish the environmental fate of protein additives in consumer products, it is necessary to be able 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 ultimate goal is to link molecular analysis directly to molecular environment. Much like a forensics officer tasked with determining the presence of an illicit substance, there is much greater reliability and credibility afforded to an analysis performed at the scene of the crime than to one performed following removal of the sample to a separate location and alternative surroundings. Growing evidence suggests in situ protein analysis has groundbreaking roles to play in biomarker discovery, diagnosis & early detection of disease, targeting therapeutics (personalised medicine) and assessment of therapeutic efficacy. The benefits of in situ protein analysis can be illustrated by considering a thin tissue section through a drug-treated tumour. In principle, in situ analysis would inform on drug-target interactions (i.e., is the drug binding to the correct protein?). Moreover, with in situ protein analysis the capacity for artefact introduction as a result of sample preparation (e.g., application of a matrix) or sample damage is eliminated. Nevertheless, a number of challenges exist. Proteins are large molecules associated with a vast array of chemical modifications, and which form loosely-bound complexes with themselves, other proteins and other molecule types. It is not only their chemical structure but also their overall 3-D structure which dictate their function. Other molecular classes that are hugely important in biological processes also have an intricate relationship with proteins. Any in situ mass spectrometry approach needs to be able to meet these analyte-driven challenges, i.e., it must be capable of (a) measuring proteins and characterising any modifications, (b) detecting protein complexes and determining their constituents, (c) providing information on 3-D structure, and (d) detecting other relevant molecular classes. Moreover, there are technique-driven challenges for in situ analysis including inherently high sample complexity and wide ranging concentrations, and opportunities for quantitation. The research will meet these challenges by developing a newly emerging in situ approach, liquid extraction surface analysis mass spectrometry, in combination with two complementary types of ion mobility spectrometry (which can either provide information on 3-D structure, or separate ionised molecules in the mass spectrometer on the basis of their 3-D shape) and a structural elucidation strategy known as electron-mediated dissociation mass spectrometry. The research will be undertaken primarily at the University of Birmingham in the Advanced Mass Spectrometry Facility in the School of Biosciences and the School of Chemistry mass spectrometry facility. The programme involves a number of academic and industrial collaborators and additional research will be carried out during scientific visits to National Physical Laboratory (NPL), Thermo Fisher Scientific, Waters, Owlstone, Florida State University, Texas A&M University and Université d'Aix-Marseille.