... understanding life in molecular detail

Prof Alison Ashcroft

Mass spectrometry, Ion Mobility Spectrometry, Biomolecular analysis, protein folding and assembly


My group develops and applies new mass spectrometry methodologies to the analysis of the structure and functional behaviour of biomolecules and biomolecular complexes.

Current major projects include:
  • protein aggregation e.g. amyloid fibril assembly
  • mapping virus capsid and baseplate assembly pathways
  • membrane protein analysis
  • mass spectrometry method development

Introduction

Figure 1: ESI-IMS-MS

Prof. Ashcroft’s research is focused on developing and applying mass spectrometric techniques and methodologies to the analysis of biomolecules. Much of our work uses electrospray ionisation (ESI) non-covalent mass spectrometry (MS) coupled with ion mobility spectrometry (IMS) and thus we are able to measure the molecular mass and size (i.e. cross-sectional area) of individual biomolecules from within highly heterogeneous mixtures in a single experiment.

The Mass Spectrometry Facility houses five ESI-MS mass spectrometers, three equipped with IMS devices and two with HPLC facilities, and we acknowledge with gratitude funding from the BBSRC, the Wellcome Trust and Waters UK Ltd. for these instruments.

 

Protein folding

Our early ESI-IMS-MS studies on protein folding involved the separation of co-populated protein conformers and measuring their populations as the protein folds/unfolds. This work led to the Journal of the American Society for Mass Spectrometry’s “Ron Hites Award” in 2009 for “outstanding original research”.

 Figure 2: Separation of three conformers (A, B, C) of the protein beta-2-microglobulin (11,860.4 Da) using ESI-IMS-MS showing the IMS drift time vs. m/z vs. intensity.

 

Amyloid assembly

We are using ESI-IMS-MS to study protein folding/unfolding and subsequent self-assembly into amyloid fibrils (in collaboration with Prof. Sheena Radford). The analyses are carried out in real-time and so we can map the assembly pathway of the amyloidogenic protein, monitoring the individual oligomers formed during aggregation. We are also investigating small molecule inhibition of amyloid fibrils by studying the interactions of these molecules with the individual protein conformers and oligomers present.

 

Virus capsid and baseplate assembly

Virus capsids and virus baseplates are macromolecular assemblies built from protein subunits. We are using ESI-IMS-MS to monitor the conformation properties of the proteins and their assembly pathways (in collaboration with Prof. Peter Stockley and Drs Nicola Stonehouse and John Barr in Leeds, and Prof Christian Cambillau (Marseille) and Dr Neil Ferguson (UC Dublin)).

 Figure 3: Lactococcal phage TP901-1 baseplate with 12 Dit subunits (pink), 18 BppU subunits (yellow) and 54 BppL subunits (blue). Measured mass 1.769 MDa; calculated mass 1.767 MDa.

Taken from D. A. Shepherd, D. Veesler, J. Lichière, A. E. Ashcroft, C. Cambillau, Molecular & Cellular Proteomics, 10, (9) M111.009787, 2011.

 

Membrane protein analyses

Membrane proteins are challenging to analyse due to their insolubility in many solvents. We are currently developing new mass spectrometric techniques of delivering membrane proteins to the mass spectrometer whilst maintaining their native-like conformation e.g. in detergents, or amphipols (in collaboration with Prof. Jean-Luc Popot, Paris) in order to study their folding and function (in collaboration with Profs. Peter Henderson and Stephen Baldwin).

Detailed research programme                  Close ▲
AEA.jpg

Professor of Biomolecular Mass Spectrometry
BSc (Liverpool) PhD (Manchester)
J. Amer. Soc. Mass Spectrom. Ron Hites Award (2009) for "outstanding original research"; H. E. Potts Medal Award (2014) for "Outstanding Contribution to Chemistry" University of Liverpool; RSC Rita & John Cornforth Award (2015) (with S. E. Radford) for "seminal contributions in mass spectrometry to characterise protein mis-folding mechanisms and inform on new strategies to combat human disease

Post-doctoral Research Fellow (University of Geneva, Switzerland) 1981
Applications & Development Mass Spectrometrist (Kratos Analytical) 198
Senior Mass Spectrometrist: Drug Metabolism (AstraZeneca Pharmaceutica
Senior Mass Spectrometrist (Micromass/Waters) 1991-1997

Astbury Building, 8.110
School of Molecular and Cellular Biology
0113 343 7273
a.e.ashcroft@leeds.ac.uk
http://www.personal.leeds.ac.uk/~fbsmaspe/research.html

Selected Publications

  1. Screening and characterising small molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry, L. M. Young, J. C. Saunders, R. A. Mahood, C. H. Revill, R. J. Foster, L. H. Tu, D. P. Raleigh, S. E. Radford, A. E. Ashcroft, Nat. Chem., 7, (1), 73-81, 2015. doi: 10.1038/NCHEM.2129.

  2. Changes in protein structure monitored by use of gas phase hydrogen-deuterium exchange, H. S. Beeston, J. R. Ault, S. D. Pringle, J. M. Brown, A. E. Ashcroft, Proteomics, “Proteomics in Protein Structure", 15, (16), 2842-2850, 2015. doi: 10.1002/pmic.201400440.
     

  3. Amphipols outperform dodecylmaltoside micelles in stabilizing membrane protein structure in the gas phase, A. N. Calabrese, T. W. Watkinson, P. J. F. Henderson, S. E. Radford, A. E. Ashcroft, Anal. Chem., 87, (2), 1118–1126, 2015. doi: 10.1021/ac5037022.

  4. Using ion mobility spectrometry-mass spectrometry to decipher the conformational and assembly characteristics of the hepatitis B capsid protein, D. A. Shepherd, K. Holmes, D. J. Rowlands, N. J. Stonehouse, A. E. Ashcroft, Biophysical J. 105, 1258-1267, 2013. doi: 10.1016/j.bpj.2013.07.028.