Characterisation of amyloid assembly using mass spectrometry

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology

Abstract

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.

Technical Summary

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.

Publications

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Ramirez-Alvarado, Marina; Kelly, Jeffery W.; Dobson, Christopher M. (2010) Protein Misfolding Diseases: Current and Emerging Principles and Therapies

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Smith DP (2009) Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. in European journal of mass spectrometry (Chichester, England)

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Smith DP (2010) Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. in Proceedings of the National Academy of Sciences of the United States of America

 
Description In terms of amyloid formation, our specific objectives for this grant were as follows:

(i). determine the conformational dynamics of amyloidogenic proteins and the role of rarely populated unfolded states in protein assembly; this has been investigated using a range of MS techniques supported by other biophysical methods (including AUC, NMR) and chemical tests (e.g., Thioflavin-T). We have successfully employed HDX-MS, TWIMS-MS, and cross-sectional area measurements to monitor monomeric _2-microglobulin folding/unfolding and thus have highlighted and quantified three distinct conformational families (folded, partially unfolded, unfolded). The relationships between these conformers and the amyloidogenicity of the wild-type protein and several variants have been assessed [1], [2], [3]: of significant interest are the differences in behaviour between native _2-microglobulin in its free and MHC-1 bound states, and the observation of an unexpected unfolding event at neutral pH which suggests the first step in self-assembly [3].



(ii). assess the structural characteristics of oligomeric species formed early in assembly; the early oligomers (dimer, trimer, tetramer) observed during self-assembly have been probed extensively under acidic, fibril-forming conditions. The relative proportions of the oligomers during assembly, together with their stabilities and dynamics, have been assessed using ESI-TWIMS-MS, MS/MS and stable-isotope chase experiments [4]. From cross-sectional areas the physical shapes can be modeled and, in the case of trimer, multiple conformers with different characteristics have been detected [4]. These results have been compared with the completely different oligomer populations at pH 3.6 (second manuscript in preparation).



(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; our initial experiments in this area using HDX-MS to monitor protein unfolding events has indicated that some additives accelerate (e.g., copper ions, SDS) whilst others decelerate (e.g., trehalose) HDX propensity, and a manuscript is in preparation. These results will be included in the first manuscript in preparation.



(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 have successfully used cross-linking methods to isolate oligomeric structures in the case of the A_ peptide, which will be invaluable for future studies [5], and are currently developing methods for the reproducible generation of fibrils from the STYVIE peptides proposed in the grant application.



In summary, this is the first time such a comprehensive study has been achieved on individual conformers and oligomers from a heterogeneous ensemble in real-time during the early stages of amyloid formation. The project has involved the development of new technology and methods, including computing algorithms, biophysical techniques, protein engineering and stable isotope labelling experiments. Significant advances have been made into defining _2-microglobulin conformers and oligomers during the early stages of amyloidosis.
Exploitation Route 1. testing for proteins that may have amyloidogenic properties.

2. screening for potential inhibitors of amyloid fibril formation i.e. drug candidates.
Sectors Other

 
Description We have carried out further research in this are and have exctended our results to the aggregation pathways and inhibition of other amyloid proteins (Abeta, IAPP, alpha-synuclein, Ataxin-3)
First Year Of Impact 2007
Sector Pharmaceuticals and Medical Biotechnology,Other
Impact Types Societal

 
Description Defining the mechanism of small molecule inhibition of amyloid fibril formation using ion mobility spectrometry-mass spectrometry
Amount £91 (GBP)
Funding ID BB/I015361/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2011 
End 09/2015
 
Description Defining the mechanisms of aberrant protein self-assembly in protein biopharmaceuticals using ESI-MS
Amount £92 (GBP)
Funding ID BB/J011819/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2012 
End 09/2016
 
Description Structure and dynamics of oligomeric intermediates in amyloid assembly
Amount £417,000 (GBP)
Funding ID BB/H024875/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2011 
End 06/2014
 
Description collaboration 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution The development of methods for characterising amyloid fibril formaiton from protein monomers through oligomers to fibrils has led to several further collaborations: 1. Protein Crystallography Group, Instituto de Biologia Molecular e Celular - IBMC, Rua do Campo Alegre, 823 4150-180 Porto, Portugal. Preliminary data on ataxin-3 amyloid assembly; one publication; one manuscript in preparation; about to write an EU grant proposal. 2. MRC/King's College London. Preliminary data on ubiquitinylation of ataxin-3; submitted a BBSRC research grant application in January 2014. 3. Stoneybrook University, New York, USA. Recent research on IAPP amyloid assembly and inhibition has led to two publications. 4. MedImmune, Cambridge, UK. Protein aggregation work in this grant has led to interest from Medimmune who are sponsoring a BBSRC CASE PHD student to study the problem of antibody aggregation in manufacturing.
Start Year 2011
 
Description collaboration 
Organisation MedImmune
Country United States 
Sector Private 
PI Contribution The development of methods for characterising amyloid fibril formaiton from protein monomers through oligomers to fibrils has led to several further collaborations: 1. Protein Crystallography Group, Instituto de Biologia Molecular e Celular - IBMC, Rua do Campo Alegre, 823 4150-180 Porto, Portugal. Preliminary data on ataxin-3 amyloid assembly; one publication; one manuscript in preparation; about to write an EU grant proposal. 2. Annalisa Pastore, MRC/King's College London. Preliminary data on ubiquitinylation of ataxin-3; submitted a BBSRC research grant application in January 2014. 3. Daniel Raleigh, Stoneybrook University, New York, USA. Recent research on IAPP amyloid assembly and inhibition has led to two publications. 4. MedImmune, Cambridge, UK. Protein aggregation work in this grant has led to interest from Medimmune who are sponsoring a BBSRC CASE PHD student to study the problem of antibody aggregation in manufacturing.
Start Year 2011
 
Description collaboration 
Organisation Stony Brook University
Country United States 
Sector Academic/University 
PI Contribution The development of methods for characterising amyloid fibril formaiton from protein monomers through oligomers to fibrils has led to several further collaborations: 1. Protein Crystallography Group, Instituto de Biologia Molecular e Celular - IBMC, Rua do Campo Alegre, 823 4150-180 Porto, Portugal. Preliminary data on ataxin-3 amyloid assembly; one publication; one manuscript in preparation; about to write an EU grant proposal. 2. Annalisa Pastore, MRC/King's College London. Preliminary data on ubiquitinylation of ataxin-3; submitted a BBSRC research grant application in January 2014. 3. Stoneybrook University, New York, USA. Recent research on IAPP amyloid assembly and inhibition has led to two publications. 4. MedImmune, Cambridge, UK. Protein aggregation work in this grant has led to interest from Medimmune who are sponsoring a BBSRC CASE PHD student to study the problem of antibody aggregation in manufacturing.
Start Year 2011
 
Description collaboration 
Organisation University of Leeds
Department School of Molecular & Cellular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution The development of methods for characterising amyloid fibril formaiton from protein monomers through oligomers to fibrils has led to several further collaborations: 1. Protein Crystallography Group, Instituto de Biologia Molecular e Celular - IBMC, Rua do Campo Alegre, 823 4150-180 Porto, Portugal. Preliminary data on ataxin-3 amyloid assembly; one publication; one manuscript in preparation; about to write an EU grant proposal. 2. Annalisa Pastore, MRC/King's College London. Preliminary data on ubiquitinylation of ataxin-3; submitted a BBSRC research grant application in January 2014. 3. Daniel Raleigh, Stoneybrook University, New York, USA. Recent research on IAPP amyloid assembly and inhibition has led to two publications. 4. MedImmune, Cambridge, UK. Protein aggregation work in this grant has led to interest from Medimmune who are sponsoring a BBSRC CASE PHD student to study the problem of antibody aggregation in manufacturing.
Start Year 2011
 
Description Celebrating Women in Leeds 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Annual news bulletin and ceremony to celebrate successful female staff members at the University of Leeds A.A.recognised in 2012, S.R. in 2013 Exhibition and invited speaker. Photos released, exhibited and printed in the University of Leeds' staff magazine "The Reporter".

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Participants in your research or patient groups
Results and Impact On receiving the Journal of the American Society for Mass Spectrometry's 2009 Ron Hites Award , press releases from both the University of Leeds and Waters Corpn. were announced. press release

no actual impacts realised to date
Year(s) Of Engagement Activity 2010