Enhanced Mass Spectrometry Facilities for the Astbury Centre for Structural Molecular Biology

Mass spectrometry is an analytical technique whereby biological samples such as peptides, proteins and nucleotides can be mass measured. Even fragile macromolecular complexes consisting of multiple protein molecules and other species such as RNA or DNA molecules which are held together by weak, non-covalent interactions, can be preserved and mass measured, if carefully controlled conditions are chosen. Some mass spectrometers also allow fragmentation of the sample to take place during the analysis. This generates a number of fragments each of which is mass measured. The results from these fragments can then be stitched together, like pieces of a jigsaw puzzle, to give a picture of the structural composition of the sample. This is particularly useful for peptides, whose amino acid sequence can be determined in this way. We propose to replace two of our old mass spectrometers with a state-of-the-art instrument that will enable us to both measure masses and carry out structural determinations. This equipment is vital for our research, which covers many aspects of structural molecular biology. One project involves an investigation into the RNA-triggered assembly of virus capsids from protein monomers. By studying this reaction and characterising key intermediates on the way to the final virus capsid, we can seek to understand, and subsequently devise methods to prevent, virus assembly. Another project looks into the way certain proteins fold into their unique 3D shape. If a protein unfolds, there is an opportunity for it to fold incorrectly. This phenomenon is thought to occur in vivo, where some ~20 proteins misfold and aggregate into large, insoluble, polymeric complexes which are associated with many incurable diseases such as Alzheimer's, CJD, and BSE. Other projects we are carrying out include studying the structure and behaviour of proteins and peptides from different organisms such as the mosquito and fruit fly, as well as plants, in order to determine how these species function. Peptide sequencing will be vital for these studies. The new mass spectrometer will be used by many people in the Astbury Centre for Structural Molecular Biology and the Faculty of Biological Sciences, as well as our collaborators in the Schools of Chemistry, Physics, Medicine and Dentistry: thus it will bring together many scientific disciplines. Not only will it enhance our research, which is aimed directly at understanding how biological systems work in order to improve the quality of life, it will also be used to train post-graduate and post-doctoral research workers from many groups, and will provide us with an opportunity to develop new mass spectrometric techniques that we will apply to novel bioanalyses and these will be beneficial to many other people in academia and in industry.

Mass Spectrometry has emerged over the past 17 years as a powerful technique that can be applied successfully to a variety of biological applications, not simply as a mass detector but also as a tool for peptide sequencing and associated proteomics studies, as a means of monitoring reactions such as protein folding and aggregation in real time, for determining the stoichiometry of non-covalently bound complexes and for assessing the strength of protein-ligand binding interactions. Mass Spectrometry is of vital importance to the Astbury Centre of Structural Molecular Biology and the Faculty of Biological Sciences at the University of Leeds. Not only does the Mass Spectrometry Facility support cutting-edge research and undertake development work to extend our portfolio of biological applications, it also carries out a fast and reliable service providing mass measurements and structural information. Two of our existing mass spectrometers have been declared 'obsolete' by the manufacturer and need to be replaced as a matter of urgency. We have identified an instrument that can replace these two mass spectrometers, whose key features are high sensitivity, a high m/z range, a high MS/MS m/z range and a novel design incorporating a Travelling Wave Ion Guide. The new instrument will cover our service work and will also enhance fundamental MS research allowing new experiments including in situ dissociation of large complexes. Additionally, the Travelling Wave Ion Guide will be used as an Ion Mobility Spectrometry device and thus add a further dimension to our research by allowing the development of methods to separate and measure the mass and size of co-populated protein conformers, macromolecular complexes and key assembly intermediates. Our research focus has many well-funded projects including (i) the assembly pathway of virus capsids; (ii) protein folding, misfolding and amyloid disease; (iii) membrane protein structure; (iv) pilus biogenesis; (v) proteomics.