The analytical technique of ion mobility spectrometry was developed by Cohen and Karasek in 1970 as a sensor building on earlier gas-phase ion chemistry investigations. It has since been used to detect a wide range of analytes including illegal drugs, chemical warfare agents, explosives and environmental pollutants. Ion mobility is a measure of how quickly a gas phase ion moves through a buffer gas under the influence of an electric field, and this depends on two factors: the rotationally averaged collision cross section of the ion and the charge present on it. By measuring the drift time of an ion through a known distance it is possible to determine its collision cross section with some degree of accuracy. In instruments where the drift field is a dc potential, the relationship between the collision cross section of an ion and the measured average drift time can be easily found. The experimental collision cross section can be compared to cross sections predicted from co-ordinates obtained from other structural investigations, or from computational measurements to obtain atomistically detailed conformational information. The relationship between the drift times of ions in a ion mobility spectrometer and their gas-phase collision cross section, is well understood, and in recent years ion mobility spectrometry coupled with mass spectrometry (IM-MS) has gained particular importance as a tool for structural analysis and particularly for its use to reveal conformation of biological molecules. After developments in soft ionization methods, IM-MS studies of biological relevant species started in the mid and late 1990's on home built instruments which coupled these two well known analytical techniques. Some of the most influential work in this period was performed by Bowers, Jarrold, Clemmer, and Hill and their investigations have paved the way for others and prompted development of commercially available mobility devices, as the power of this technique for biological analysis became apparent. Waters MS Technologies (Manchester, UK) recently introduced the first commercially available integrated IM-MS instrument the Synapt HDMS. The RF applied to consecutive electrodes in the stacked ring ion guide within the ion mobility separator, provides a potential well which keeps the ions radially confined within the device. In order to propel the ions through the device, a travelling wave comprising a series of transient DC voltages is superimposed on top of the RF voltage, and hence this device is sometimes referred to as a Travelling Wave Ion Guide - TWIG. This voltage is applied sequentially to pairs of ring electrodes providing a potential which can push ions through the device. These commercial available devices have already been used to good effect. Using a TWIG based system, Robinson et al. have assessed conformations of multimeric proteins, and also the disassembly of complexes viewing the partial unfolding of monomer units whilst still retaining some the integrity of the complex. The benefits of the Synapt compared to home built instruments are undisputed, the duty cycles are shorter and the transmission efficiency through this instrument is better than with most home made devices. However, to properly rationalize experimental drift times obtained on Synapt instrumentations in terms of collision cross sections, requires careful calibration with data obtained on a linear ion mobility instrument, such as that developed by Bowers and Clemmer and also present in the lab of Barran. One of the issues with this is that the available collision cross section data for proteins is limited, and also often not well verified. This means that despite the extreme interest in the application of the Synapt to interrogate complex biological structures, and beautiful preliminary work, results are still somewhat 'unverified' This studentship will seek to address this in several ways. See the Research Strategy below.

Lipids are the fatty molecules that make up the membranes that surround cells; without them life would not exist. However, this is only one of the important roles that lipids play in biology. For example, lipids are also involved in many forms of communication within and between cells, and the lipid composition of the cell membrane affects the activity of proteins embedded in it, such as those that transport molecules in and out of the cell. Damage to lipids by reaction with oxygen, in the same way that cooking oils go rancid, is related to many of their roles in disease. Hence, the analysis of lipids and understanding their roles in biology are very important areas of research. However, the comprehensive analysis of lipids is challenging as they are a very complex set of molecules, with over 100,000 different types in human cells, and many more when bacteria and other microorganisms are included. Many of them have very similar chemical structures, making it hard to tell them apart, but very diverse effects, so it is important to identify them correctly. The equipment we will buy with this grant has an extra dimension for the separation of molecules, based on their shape, which will greatly enhance the number of different lipids that we are able to distinguish and will enable a wide range of research to help understand their complex roles in biology. There are many examples of how lipids are important in life. For example, they play a role in controlling cell growth to cell death, including processes particularly important in conditions such as inflammation. Lipids can also affect the activity of proteins in the cell, and particularly those in the cell membranes, many of which are targets for drugs such as morphine and insulin. Analysis of the lipids associated with membrane proteins, and the effects that changing of these lipids has on the activity of the proteins, is important in understanding these effects and how they may change with age or diet, or in other diverse areas, such as the production of biofuels or processes in bacterial replication that could be new targets for antimicrobials. Lipids contain many sites that can be attacked by reactive chemical species, and these damaged lipids can themselves have biological activity or react with other molecules impairing their function. An example is LDL, or bad cholesterol. Oxidative damage to the lipids in LDL is thought to be responsible for changes that lead to heart attacks and strokes. We need to be able to analyse the different lipids that are generated in these reactions, how they interact with or react with biological systems, and what effects this has on the biological system. This grant proposal is provide instrumentation that will allow us to perform the complex analysis required to confidently identify and measure the amount of lipids that are present in complex biological samples. The main technique to be used is mass spectrometry, which measures the weight of molecules very accurately, as well as being able to break up the molecules to get information on their structure. However, the current methods are not always able to separate all the individual components in complex mixtures to allow their full analysis, especially of low abundance molecules that affect cell behaviour. The new instrument will provide extra capabilities through an additional dimension for separation of the molecules, called ion mobility, which is able to separate molecule based on their shape. The equipment will be the first available of a new design of instrument that allows much finer separation of molecules (it has a cyclic ion mobility cell providing much longer effective separation path lengths). This will allow us to do more accurate measurement of the lipids present and the way in which they are changed, leading to a much better understanding of biology in many important areas.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at www.rcuk.ac.uk/StudentshipTerminology. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

The project is aimed at the development of new tools for the identifications of various microorganisms including bacteria causing a wide range of diseases from common cold to bloodstream infections. Knowing exactly which type of bacterium is involved in a disease is very important, since the choice of appropriate medication largely depends on it. Likewise, in case of public health or food safety, the correct classification of bacterial contaminations helps with the identification of their source and elimination of the contamination. Currently, samples containing bacterial cells are collected and sent to laboratories. Microbiologists grow the bacteria in Petri-dishes containing special nutrients. Based on the types of nutrients the bacteria can use and the results of multiple chemical tests, the bacterium is tentatively identified. If proper classification is necessary, nucleic acids are extracted from the bacterial cells, and their base-pair sequence is determined, which helps in the unambiguous identification. All of these processes are time consuming, which considerably delays the efficient intervention both in case of infectious diseases and in case of a waterborne disease outbreak. The purpose of the proposed research is to develop an alternative, much faster technique for the identification of bacteria. Mass spectrometry is an analytical technique capable of the measurement of the weight of molecules, and also the selective detection of hundreds of different molecules at the same time. We plan to use this well-established technique for looking at some special building blocks of bacterial cells. One novel aspect of the research is that mass spectrometers are not used in the traditional way, including a lengthy preparation of bacterial cells prior to analysis, but the cells are simply heated up, and electrically charged molecules formed on the boiling of cells are analysed using mass spectrometry. The idea of rapid mass spectrometric analysis by using simply heat has already been applied in case of surgery, where cancer tissue is identified in a similar way. In course of the proposed project we plan the adopt this technology (Rapid Evaporative Ionization Mass spectrometry; REIMS) for the analysis of bacteria grown in the laboratory and also for the direct analysis of liquid samples (ranging from pond water to blood) containing bacterial agents. We plan to build a large library of the spectroscopic fingerprints of the bacteria, which will be used as a training set for computer based search algorithms. The method, the database and the algorithm together will enable the unambiguous identification of bacteria in considerably shorter timeframe than the current routine. Furthermore, the proposed research can potentially lead to an approach, where bacteria are directly identified in their natural environment (e.g. in urine for a urinary infection) without growing them in the laboratory for several hours or days.

Amyloidosis is a pathological condition associated with the self-aggregation of proteins into highly ordered amyloid fibrils in vivo. Despite the importance of these high-profile disorders in today's ageing population and a wealth of on-going research, knowledge of the structural molecular mechanism of amyloid formation remains limited, primarily because of the complexity and heterogeneity within these systems, and rationally designed therapies are rare. Several small molecules have recently been found to modulate (stimulate or inhibit) the rate of fibrillogenesis of various amyloidogenic proteins in vitro but, in many cases, their reaction mechanism remains unknown. We have recently developed novel mass spectrometric methods to separate protein conformers and oligomers in real-time. Mass, kinetic and thermodynamic stability, and shape/cross-sectional area of each component within a heterogeneous assembly reaction can be measured in a single experiment using travelling wave ion mobility spectrometry coupled to mass spectrometry (IMS-MS; engineered by Micromass/Waters). Our data demonstrate the immense power of IMS-MS to resolve transiently populated species during the early stages of amyloidosis in vitro and form the basis for this project (Smith et al., J. Am. Soc. Mass Spectrom., 2007; Smith et al., PNAS, 2010). The aim of this project is to define amyloidogenic protein-ligand binding events in detail to pave the way to the rational design of ligands able to inhibit fibrillogenesis. To achieve this we will: (i) assess changes in the protein population (i.e. ratio of protein conformers, population of oligomers, etc.) caused by ligand presence; (ii) define which species bind the ligand; (iii) compare the conformational properties of the protein monomer and oligomers pre- and post-ligand binding; (iv) monitor the effect of ligand binding on the progress of fibril assembly. The student will initially study the protein-ligand binding characteristics of beta2-microglobulin, an amyloid-forming protein with which Ashcroft and Radford have gained much experience. Specifically, the student will study the rifamycin family of small molecule macrocycles, some of which we have found inhibit amyloid fibril formation whilst others have no effect. Using IMS-MS to analyse the mixture of protein conformers and oligomers, we will assess the changes in protein population resulting from ligand presence and identify ligand-binding species. Protein-ligand binding stability will be assessed from their binding constants and by MS/MS collision induced dissociation. To detect any conformational change on binding, the shape/cross-sectional areas of pre- and post-ligand binding protein species will be measured by IMS-MS. The protein regions involved in ligand binding will also be explored using novel HDX-IMS-MS methodology in collaboration with Micromass/Waters. The MS experiments will be complemented by other biophysical measurements (size exclusion chromatography, analytical ultracentrifugation) and the presence of fibrils in each experiment will be confirmed by electron microscopy. Protein mutants with different fibril-forming propensities which have been engineered by Radford will be compared to identify ligand binding residues. Non-amyloidogenic murine beta2-microglobulin will be used as a control. After establishing robust methods, the project will widen to encompass other proteins associated with amyloid diseases. Using the techniques described, their ligand binding properties will be investigated with known inhibitors. This will include the interactions of alpha-synuclein (Parkinson's disease) with baicalein and other flavanoids; Abeta (Alzhiemer's disease) with catechol derivatives; IAPP (type II diabetes) with resveratol. Thus, a correlation between ligand binding, protein conformation and fibril formation will be assessed which will ultimately pave the way for the rational design and screening of amyloid inhibition ligands.