Enhanced Mass Spectrometry Facilities for the Astbury Centre for Structural Molecular Biology
Lead Research Organisation:
University of Leeds
Department Name: Inst of Molecular & Cellular Biology
Abstract
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.
Technical Summary
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.
Publications
Alexander CG
(2013)
Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein.
in Proceedings of the National Academy of Sciences of the United States of America
Ariza A
(2013)
Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization.
in Nucleic acids research
Bravo JPK
(2018)
Stability of local secondary structure determines selectivity of viral RNA chaperones.
in Nucleic acids research
Byrd EJ
(2023)
Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of a-Synuclein.
in Journal of the American Society for Mass Spectrometry
Calabrese A
(2014)
Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase
in Analytical Chemistry
Calabrese AN
(2015)
Using hydroxyl radical footprinting to explore the free energy landscape of protein folding.
in Methods (San Diego, Calif.)
Cornwell O
(2018)
Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and ?N6 ß2-Microglobulin.
in Journal of the American Society for Mass Spectrometry
Cornwell O
(2021)
Investigation of D76N ß2-Microglobulin Using Protein Footprinting and Structural Mass Spectrometry.
in Journal of the American Society for Mass Spectrometry
Devine PWA
(2017)
Investigating the Structural Compaction of Biomolecules Upon Transition to the Gas-Phase Using ESI-TWIMS-MS.
in Journal of the American Society for Mass Spectrometry
Dobson CL
(2016)
Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.
in Scientific reports
Doherty CPA
(2018)
A peptide-display protein scaffold to facilitate single molecule force studies of aggregation-prone peptides.
in Protein science : a publication of the Protein Society
Giusti F
(2014)
Synthesis, Characterization and Applications of a Perdeuterated Amphipol
in The Journal of Membrane Biology
Goulet A
(2011)
The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages.
in The Journal of biological chemistry
Holmes K
(2015)
Assembly Pathway of Hepatitis B Core Virus-like Particles from Genetically Fused Dimers.
in The Journal of biological chemistry
Humes JR
(2019)
The Role of SurA PPIase Domains in Preventing Aggregation of the Outer-Membrane Proteins tOmpA and OmpT.
in Journal of molecular biology
Iadanza MG
(2016)
Lateral opening in the intact ß-barrel assembly machinery captured by cryo-EM.
in Nature communications
Jürgens MC
(2013)
The hepatitis B virus preS1 domain hijacks host trafficking proteins by motif mimicry.
in Nature chemical biology
Kang L
(2012)
N-terminal acetylation of a-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer.
in Protein science : a publication of the Protein Society
Karamanos TK
(2019)
Structural mapping of oligomeric intermediates in an amyloid assembly pathway.
in eLife
Knapman T
(2010)
Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry
in Rapid Communications in Mass Spectrometry
Knapman TW
(2013)
Ion Mobility Spectrometry-Mass Spectrometry of Intrinsically Unfolded Proteins: Trying to Put Order into Disorder.
in Current analytical chemistry
Knight PD
(2018)
Identification of a novel site of interaction between ataxin-3 and the amyloid aggregation inhibitor polyglutamine binding peptide 1.
in European journal of mass spectrometry (Chichester, England)
Leney A
(2014)
Insights into the role of the beta-2 microglobulin D-strand in amyloid propensity revealed by mass spectrometry
in Mol. BioSyst.
Leney AC
(2014)
Insights into the role of the beta-2 microglobulin D-strand in amyloid propensity revealed by mass spectrometry.
in Molecular bioSystems
Leney AC
(2012)
Amphipathic polymers enable the study of functional membrane proteins in the gas phase.
in Analytical chemistry
Martin EM
(2018)
Conformational flexibility within the nascent polypeptide-associated complex enables its interactions with structurally diverse client proteins.
in The Journal of biological chemistry
Morrissey B
(2012)
The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS.
in Molecular & cellular proteomics : MCP
Nahass GR
(2021)
Brazilin Removes Toxic Alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium.
in Journal of molecular biology
Preston GW
(2014)
Analysis of amyloid nanostructures using photo-cross-linking: in situ comparison of three widely used photo-cross-linkers.
in ACS chemical biology
Preston GW
(2012)
Covalent cross-linking within supramolecular peptide structures.
in Analytical chemistry
Pritchard C
(2013)
The role of ion mobility spectrometry-mass spectrometry in the analysis of protein reference standards.
in Analytical chemistry
Rajabi K
(2015)
Mass spectrometric methods to analyze the structural organization of macromolecular complexes.
in Methods (San Diego, Calif.)
Rajabi K
(2015)
A comparison of the folding characteristics of free and ribosome-tethered polypeptide chains using limited proteolysis and mass spectrometry.
in Protein science : a publication of the Protein Society
Saunders JC
(2016)
An in vivo platform for identifying inhibitors of protein aggregation.
in Nature chemical biology
Scarff C
(2013)
A tale of a tail: Structural insights into the conformational properties of the polyglutamine protein ataxin-3
in International Journal of Mass Spectrometry
Scarff CA
(2015)
Examination of Ataxin-3 (atx-3) Aggregation by Structural Mass Spectrometry Techniques: A Rationale for Expedited Aggregation upon Polyglutamine (polyQ) Expansion.
in Molecular & cellular proteomics : MCP
Scarff CA
(2016)
Characterization of Amyloid Oligomers by Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry (ESI-IMS-MS).
in Methods in molecular biology (Clifton, N.J.)
Schiffrin B
(2016)
Skp is a multivalent chaperone of outer-membrane proteins.
in Nature structural & molecular biology
Schiffrin B
(2017)
Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding.
in Journal of molecular biology
Shepherd DA
(2014)
Probing bunyavirus N protein oligomerisation using mass spectrometry.
in Rapid communications in mass spectrometry : RCM
Shepherd DA
(2013)
Using ion mobility spectrometry-mass spectrometry to decipher the conformational and assembly characteristics of the hepatitis B capsid protein.
in Biophysical journal
Shepherd DA
(2011)
Unraveling lactococcal phage baseplate assembly by mass spectrometry.
in Molecular & cellular proteomics : MCP
Smith DP
(2009)
Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies.
in European journal of mass spectrometry (Chichester, England)
Smith DP
(2011)
Structure and dynamics of oligomeric intermediates in ß2-microglobulin self-assembly.
in Biophysical journal
Smith DP
(2008)
Trifluoromethyldiazirine: an effective photo-induced cross-linking probe for exploring amyloid formation.
in Chemical communications (Cambridge, England)
Smith DP
(2007)
Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry.
in Journal of the American Society for Mass Spectrometry
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 | the mass spectrometer has been used every day for the analysis of proteins. we have developed new methods to measure both the mass and cross-sectional area of the protein in a single experiment. we have used this for the characterisaiton of many proteins and biomolecular complex assmebly pathways. |
Exploitation Route | by reading our extensive literature |
Sectors | Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology Other |
Description | the mass spectrometer has been used every day for the analysis of proteins. we have developed new methods to measure both the mass and cross-sectional area of the protein in a single experiment. we have used this for the characterisaiton of many proteins and biomolecular complex assmebly pathways. |
First Year Of Impact | 2007 |
Sector | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other |