Characterisation of amyloid assembly using mass spectrometry

Proteins consist of a chain of covalently bound amino acid units, and the order in which the different amino acids form the chain is unique to that protein. In the living cell, protein chains fold into a unique 3-dimensional configuration. Often they form non-covalent interactions with other proteins, as well as small molecules and ions within the cell, to make macromolecular biological complexes. Most of the work in cells is performed by such complexes, rather than by individual proteins working alone. It is important that we try to understand how proteins form such complexes, so that we can understand how proteins function. If a protein unfolds from its 3-dimensional structure and then does not fold back again correctly, the protein can mis-function. Some proteins misfold and then polymerise to form large, well-ordered polymers known as amyloid fibrils. These fibrils are associated with several high-profile diseases including Alzheimer's disease, Type II diabetes, haemodialysis-related amyloidosis and the prion diseases including bovine spongiform encephalopathy ('mad cow' disease) and Creutzfeldt-Jakob disease. Despite the significance of amyloid fibrils in human health, and other exciting potentials for utilising protein fibrils in a beneficial way, we currently know little about how normally soluble proteins assemble into amyloid fibrils. In this proposal, we will use mass spectrometry to elucidate new information and protein self-assembly mechanisms. This method involves injecting a protein in solution into a mass spectrometer whereupon protein molecules are ionised, using a technique known as electrospray ionisation, and then separated according to their mass-to-charge ratio. These data are recorded onto a spectrum from which the molecular mass of the protein can be determined. As well as molecular mass information however, electrospray ionisation mass spectrometry can also reveal additional details about a protein, such as whether the protein is correctly folded and whether it is in monomeric or oligomeric form. We have been studying the protein beta-2-microglobulin. In healthy humans, beta-2-microglobulin is excreted from the kidney. If a person is suffering from kidney failure and undergoing dialysis treatment, beta-2-microglobulin is not excreted, and forms insoluble fibrils that build up in the ankle, knee, hip, elbow and shoulder joints. This disease, known as haemodialysis-related amyloidosis, causes much pain and eventually is fatal. We have already developed methods to quantify a mixture of folded, partially folded and unfolded beta-2-microglobulin molecules; we have also monitored the aggregation of the protein monomer and seen the dimer, trimer, tetramer, etc appear and disappear during fibril formation. Also we have developed methods to look at the structure of the fibrils by using enzymes to remove accessible protein fragments from the fibrils, and then characterising these fragments by mass spectrometry. Here we propose to examine beta-2-microglobulin in its natural environment, i.e. surrounded by biological species and at physiological pH, to discover how the protein might self-assemble in vivo. We will also develop new mass spectrometry methods to decipher more precisely the aggregation reaction mechanism and structure of the fibrils formed.

Protein misfolding and self-assembly gives rise to structurally beautiful, but deadly, amyloid fibrils: a phenomenon that is associated with the onset of ca. 20 different human diseases. Despite the importance of this biological self-assembly process, the molecular mechanisms by which proteins self-assemble into amyloid fibrils remains unsolved. Electrospray Ionisation (ESI) - Mass Spectrometry (MS) is a highly sensitive and versatile technique that not only produces accurate mass measurements of proteins, but also provides information regarding a protein's physical state and behaviour in solution. Our objective is to exploit the full potentials of MS and to develop innovative MS methods, to provide new insights into amyloid assembly mechanisms. Specifically, we will: (i). determine the conformational dynamics of amyloidogenic proteins and the role of rarely populated unfolded states in protein assembly; (ii). assess the structural characteristics of oligomeric species formed early in assembly; (iii). examine the role in protein assembly of glycosaminoglycans, SAP and ApoE. These factors are known to be associated with all amyloid diseases, but little is known about the molecular recognition event between these factors and assembling proteins. Specifically we will use MS to identify the target substrates for these ligands (monomer, oligomer or fibril) as well as the nature of their molecular recognition event and the effect of these factors in assembly; (iv). test models of nucleation for a range of small simple peptide assemblies, so that generic principles about the early events in assembly can be discerned. We will address these questions by exploiting the full power of modern ESI-MS methods as well as by developing new MS methods, using the amyloidogenic protein beta-2-microglobulin as a platform for this research. The methods used will include: (a). deconvolution of ESI-MS charge state distributions to study conformer populations co-populated in solution; (b). development of ESI-high Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS)-MS to uncover rare protein conformers; (c). characterisation of oligomeric assembly intermediates under noncovalent ESI-MS conditions; (d). analysis of oligomer and fibril structure and their binding properties using hydrogen-deuterium exchange (HDX)-MS and limited proteolysis with tandem MS/MS peptide sequencing; (e). analysis of protein conformational dynamics using real-time on-line HDX-MS.