Mass spectrometry facility

Alison E. Ashcroft

 

Overview of facility

The mass spectrometry facility (http://www.astbury.leeds.ac.uk/facil/mass.htm) has a Platform II single-quadrupole electrospray instrument with on-line HPLC capabilities, a Q-Tof orthogonal acceleration quadrupole - time of flight tandem instrument with nanospray ionisation and a TofSpec matrix assisted laser desorption ionisation mass spectrometer. The facility runs an analytical service as well as being actively involved in several research projects with groups within the Astbury Centre for Structural Molecular Biology and the Faculty of Biological Sciences, and also with external collaborators.

 

Research projects

The research involves the development of electrospray mass spectrometric techniques to aid the structural elucidation of biomolecules and can be categorised into four main areas:

i. Protein folding

Protein folding is an intriguing area in biochemistry and protein mis-folding is thought to be responsible for several diseases. Working with Prof. S. E. Radford's group, electrospray mass spectrometry is being used to monitor folding profiles and intermediates for the protein b 2-microglobulin (a 99-residue b -sheet protein) using hydrogen-deuterium exchange in order to gain an insight into folding, mis-folding and hence fibril formation.

ii. Protein - ligand non-covalent interactions

In collaboration with the groups of Prof. P. G. Stockley, Prof. S. E. Radford and Dr N. J. Stonehouse, controlled mass spectrometric conditions that preserve non-covalent interactions are being employed to investigate and characterise macromolecular structures. Such studies include protein-peptide, protein-protein, and protein-RNA complexes. The latter are particularly important in virus assembly, an area we are investigating with respect to the MS2 and Qb systems. By preserving virus assembly and disassembly intermediates in the gas phase we are using mass spectrometry to determine their composition and stoichiometry. Protein-protein macromolecular complexes are important species in fibril formation and are under investigation as an integral part of our b 2-microglobulin folding studies.

iii. Reaction monitoring

Reaction monitoring by mass spectrometry is being employed in areas such as the thrombin activation of Factor XIII A-subunit with Prof. P. J. Grant and Dr R. A. S. Ariens of the Medical School, and for monitoring enzyme-substrate formation with Dr C. W. Wharton, University of Birmingham.

Factor XIII (FXIII) is a transglutaminase that plays an important role in blood coagulation. It is converted by thrombin to its activated form which catalyses the covalent cross-linking of fibrin to increase the stability of the fibrin clot. Deficiency of FXIII results in uncontrolled bleeding, poor wound healing and miscarriage in pregnancy. Figure 1 shows the mass spectrum of FXIII A-subunit (83,192-3Da) which was mass measured to an accuracy of ± 0.01% before being treated with thrombin. Both the activated A-subunit (79,256.7 Da) and the activated peptide (3951.2 Da) were identified after proteolytic cleavage, thus substantiating the proposed amino acid sequences.

 

Figure 1: Mass spectrum of Factor XIII A-subunit, molecular weight measured 83,192.3 Da
(calculated 83, 192.4 Da).

 

iv. Structural elucidation

Tandem mass spectrometric sequencing of proteins is useful for the confirmation of amino acid sequence. This technique is being used to characterise membrane proteins, after digestion with a variety of enzymes to ensure a high sequence coverage, in collaboration with Prof. P. J .F. Henderson and Dr R. B. Herbert's group. We have also entered the field of proteomics working with external collaborators (Prof. R. A. Wilson, University of York; Dr R. E. Banks, St. James' Hospital) to generate de novo mass maps and sequence tags from 2-D gel digests.

 

References

A.M. Parrott, H. Lago, C.J. Adams, A.E. Ashcroft, N.J. Stonehouse, P.G. Stockley. 2000 "RNA Aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour". Nucleic Acids Research, 28, 2, 489-497.

S. Jones, J.S. Reader, M. Healy, A.E. Ashcroft, A.P. Kalverda, D.A. Smith, S.E. Radford. 2000 "Unfolding of the Greek Key Protein Y74W Apo-pseudoazurin: Evidence of an Equilibrium Intermediate Species". Biochemistry, 39, 5672-5682.

A.E. Ashcroft, P.J. Grant, R.A.S. Ariens. 2000 "A study of Human Coagulation Factor XIII A-subunit by Electrospray Ionisation Mass Spectrometry." Rapid Communications in Mass Spectrometry, 14, 1607-1611.

Funding

We wish to acknowledge financial support from The Wellcome Trust, BBSRC, Micromass UK Ltd and The University of Leeds.