October 14, 2010

Pushing the Boundaries of Mass Spectrometry

Manchester Interdisciplinary Biocentre (Interactive Map)

Time Speaker

Plenary Lecture
Professor Carol Robinson (Department of Chemistry, University of Oxford)
Dehydrated but unharmed? Protein Complexes in the Gas Phase

1.00 Lunch and Posters
2.00 Sabine Flitsch
Application of MALDI-ToF MS for the analysis of enzymatic reactions on gold arrays
2.25 James Allen
Time-Of-Flight Mass Spectrometry Used in Measurements of Atmospheric Aerosols and Exploring Other Applications
2.50 Jon Humphries
Proteomic analysis of the dynamics of integrin adhesion receptor signalling
3.15 Coffee and Posters
3.40 Will Allwood, Roy Goodacre
Mass spectrometry meets metabolism: how MS in Manchester has driven forward metabolomics
4.05 John Fletcher
ToF-SIMS -  Mass Spectrometry for 2 and 3D Biological Imaging
4.30 End




Dehydrated but unharmed? Protein Complexes in the Gas Phase

Carol V Robinson, Department of Chemistry, Physical and Theoretical Chemistry Laboratory, Oxford

Recent progress in proteomics has yielded vast arrays of interacting proteins together with knowledge of their post translational modifications. A key challenge is to integrate this information with homology modelling and structural biology to provide a topological, or even an atomic context for these modifications. Mass spectrometry is ideally placed to bring together these largely independent disciplines, being already established in proteomics and rapidly emerging in structural biology. By using mass spectrometry based approaches to probe intact complexes subunit stoichiometry, interaction networks and subunit packing can be defined, leading ultimately to 3D models [1]. In my lecture I will show how superimposing proteomics information and ion mobility data onto both low resolution 3D models [2] and high resolution atomic structures [3], is revealing new insight into function.

Many of these developments were made during the study of soluble complexes. Very recently we have had a significant breakthrough that has enabled us to begin studying membrane protein complexes by mass spectrometry [4].  Using examples of the intact V and F-type ATPases I will describe how using ion mobility mass spectrometry we are able to build confident 3D models of these complexes that are contributing to our growing understanding of this important class of biomolecules.  


  1. T. L. Pukala, B. T. Ruotolo, M. Zhou et al., Structure 17 (9), 1235 (2009).
  2. M. Zhou, A. M. Sandercock, C. S. Fraser et al., Proc. Natl. Acad. Sci. U.S.A. 105 (47), 18139 (2008).
  3. H. Hernandez, O. V. Makarova, E. M. Makarov et al., PloS one 4 (9), e7202 (2009).
  4. N. P. Barrera, N Di Bartolo, P. J. Booth et al., Science 321 243 (2008); N. P. Barrera, S. C. Isaacson, M. Zhou et al., Nat Methods 6 (8), 585 (2009).


Application of MALDI-ToF MS for the analysis of enzymatic reactions on gold arrays

Sabine L Flitsch, School of Chemistry & MIB, The University of Manchester

Self-assembled monolayers (SAMs) on gold have become widely used as an attractive platform for studying chemical and biochemical reactions, for studying biomolecular interactions and for the development of nanoscale devices. We have used the platform to study the solid-supported synthesis of carbohydrates and glycopeptides using both chemical and enzymatic methods. An attractive feature of the technology is the opportunity for miniaturisation and in situ analysis using mass spectrometry, SPR and fluorescence spectroscopy. Applications for the synthesis of complex oligosaccharides and glycopeptides to generate glycoarrays and their potential for disease screening will be discussed.

Laurent et al ChemBioChem (2008) 9, 883-887; Zhi et al ChemBioChem (2008) 9 (10) 1568 – 1575; Laurent et al. Trends in Biotechnology (2008) 26 328-337; Laurent et al Chem. Commun. (2008) 4371-4384; Laurent et al ‘ChemBioChem (2008) 9, 2592-2596; Deere  et al. Langmuir (2008), 24(20), 11762-1176; Haddoub et al Org. Biomol. Chem. (2009) 7, 665–670; Voglmeir et al OMICS J. (2010) in press.


Time-Of-Flight Mass Spectrometry Used in Measurements of Atmospheric Aerosols and Exploring Other Applications

James Allan,1,2 Hugh Coe,1 Douglas Worsnop,3 John Jayne,3 Joel Kimmel,3,4 Michael Cubison,4 Marc Gonin4 and Katrin Fuhrer4

1School of Earth, Atmospheric & Environmental Science, University of Manchester
National Centre for Atmospheric Science
3Aerodyne Research, Billerica, MA, USA
Tofwerk AG, Thun, Switzerland

Atmospheric particulates are important to understand, as they have major effects on climate and human health. The Aerodyne Aerosol Mass Spectrometer (AMS) is an instrument designed to study particulate composition online by using thermal vaporisation and electron ionisation. The use of a novel Tofwerk Time-Of-Flight Mass Spectrometer has offered a major improvement to the instrument over the previous quadrupole system, in terms of both sensitivity and resolution. The system will be discussed, along with the techniques that have been developed to cope with the rigours of continuous operation in harsh environments and extract usable and quantitative data products from complex datasets. This includes the numerical separation of the different organic and inorganic chemical components in the absence of chemical separation and estimation of parameters such as the O:C ratio of the organic fraction.

The Tofwerk TOF-MS is highly versatile, allowing intermediate-resolution analysis of continuous ion sources with a relatively short drift length, compact size, high dynamic range and fast rate of data acquisition. Other bespoke applications of this mass spectrometer to other atmospheric composition measurements that are currently under development by a number of other universities will be presented. These all use a common data format and we are involved in efforts to develop universally applicable data processing software that draws on our experience with the AMS.


Proteomic analysis of the dynamics of integrin adhesion receptor signalling

Jonathan D. Humphries,1,2 Adam Byron,1,2 Janet A. Askari,1,2 David Knight2 and Martin J. Humphries1,2

1Wellcome Trust Centre for Cell-Matrix Research
2Faculty of Life Sciences, University of Manchester

Adhesion to the extracellular matrix, via cell-surface integrin adhesion receptors, initiates signals that control cell morphology, movement, survival and differentiation in various developmental, homeostatic and disease processes. Integrin-ligand binding orchestrates the assembly of membrane-associated signalling complexes and mechanosensitive connections to the actin cytoskeleton. Integrin adhesion complexes readily fall apart during their isolation as cells are disrupted and have therefore been refractory to proteomic analysis. We have developed a methodology for the affinity isolation and mass spectrometric analysis of stabilised, ligand-induced integrin adhesion complexes (Humphries et al., 2009). Here, we report the cataloguing of the proteomes, at multiple time points, of complexes associated with multiple integrin receptor–ligand pairs. Hierarchical clustering of identified proteins revealed distinct temporal dynamics of protein modules relevant to cell adhesion processes. Quantitative, comparative analyses highlighted temporal profiles of integrin-associated networks that correlated with activation state–dependent signals during the initial stages of cell adhesion. The development of this proteomic workflow now allows the molecular dynamics of adhesion complexes to be measured directly and presents an entry point for quantitative, systems-level analyses of adhesion signalling in health and disease.


Mass spectrometry meets metabolism: how MS in Manchester has driven forward metabolomics

Will Allwood, Warwick Dunn, Marie Brown, Paul Begley, Eva Zelena, Husermet consortium, Douglas Kell and Roy Goodacre.

Metabolomics focuses on the study of low molecular weight organic and inorganic biochemicals in a range of biological systems including microbes, plants and mammals [1-3]. These metabolites include organic acids, sugars and lipids but in most cases not peptides or proteins. Study objectives include biotechnology and plant strain improvement, identification of biomarkers related to disease or drug toxicity/efficacy and discovery of mechanisms of disease onset/progression. As with other omics, there are generally two experimental strategies; (a) holistic profiling studies of a large proportion of metabolites where the metabolites of biological interest are derived post data acquisition (metabolic profiling) and (b) targeted and fully quantitative analysis of a limited number of metabolites of biological interest (targeted analysis).

Current limitations in metabolomics research have included reproducible and holistic profiling of metabolomes with good metabolome coverage, metabolite identification and the expansion from small-scale to large-scale studies in human-based studies. Researchers in Manchester have pioneered a range of tools to drive metabolomic research forward in these areas and which is internationally recognised. Applications in Manchester have applied chromatography coupled to mass spectrometry as the weapon of choice. Advances in Manchester have included methodological advances for data acquisition (including closed-loop optimisation[4]), methods for automated and high-throughput metabolite identification [5] and the ability to perform large-scale biological studies where n=1000s with an analytical platform not known for long-term reproducibility [6, 7].

This presentation will discuss the evolution of methodologies and instrumentation to their current status where applications involve the study of thousands of human subjects in a single study. The following will be discussed; (a) closed-loop method optimisation of mass spectrometry, (b) automated and high-throughput procedures for metabolite identification and (c) analysis of 1000s of samples in human studies with the application of quality control samples for QA and data integration.

[1] Dunn WB, Phys Biol 2008, 5(1):011001.
[2] Kell DB, FEBS J 2006, 273:873-894.
[3] Goodacre R, et al. Trends in Biotechnology, 2004, 22(5):245-252.
[4] O'Hagan S, et al. Analytical Chemistry 2005, 77(1):290-303.
[5] Brown M et al. Analyst 2009, 134(7):1322-1332.
[6] Begley P et al. Analytical Chemistry 2009, 81(16):7038-7046.
[7] Zelena Eet al. Analytical Chemistry 2009, 81(4):1357-1364.


ToF-SIMS -  Mass Spectrometry for 2 and 3D Biological Imaging

John S. Fletcher, Nicholas P. Lockyer, John C. Vickerman

Manchester Interdisciplinary Biocentre, University of Manchester

For many years secondary ion mass spectrometry (SIMS) has been applied to a multitude of samples with applications ranging from atmospheric chemistry to zeolite analysis and is used routinely in the semi-conductor industry. The strengths of the technique; the sensitivity and specificity of mass spectrometry, high surface sensitivity and high lateral resolution imaging also make SIMS a unique and powerful tool for biological analysis.

However, for many years the technique has suffered from limited sensitivity to biological molecules as prolonged analysis of the sample results in the destruction of chemical bonds and loss of molecular information with the spectrum shifting towards atomic and generic small fragment ions. This failing has been addressed by the introduction of polyatomic ion beams, particularly C60, for sample bombardment. By delivering their energy only into the upper most surface of the analyte C60 greatly ameliorates and in some cases completely removes the problem of sub-surface damage accumulation [1]. Molecular signals can be sustained under prolonged analysis thus increasing sensitivity and also, as the sample is being eroded as the analysis progresses, allowing molecular information to be monitored both laterally and as a function of depth i.e 3D molecular imaging mass spectrometry is possible [2].

Conventional ToF-SIMS instruments however are not able to fully capitalise on the abilities of these new polyatomic ion beams, the direct coupling of the ToF analyser to the secondary ion generation results in a low duty cycle and means that mass resolution is often sacrificed during high resolution imaging. Recently we have developed a new ToF-SIMS instrument that decouples the sputtering and ionisation from the mass spectrometry thus providing a 103 increase in duty cycle while maintaining high mass resolution in all modes of operation [3]. Here we present data from this instrument from the imaging of biological tissue sections and the 3D analysis of single cells.

[1] Fletcher, John S.; Lockyer, Nicholas P.; Vickerman, John C., C60, Buckminsterfullerene: its impact on biological ToF-SIMS analysis. Surface and Interface Analysis 38 (2006) 1393-1400

[2] Fletcher, John S.; Lockyer, Nicholas P.; Vaidyanathan, Seetharaman; Vickerman, John C., TOF-SIMS 3D Biomolecular Imaging of Xenopus laevis Oocytes Using Buckminsterfullerene (C60) Primary Ions. Analytical Chemistry 79 (2007) 2199-2206

[3] John S. Fletcher, Sadia Rabbani, Alex Henderson, Paul Blenkinsopp, Steve P. Thompson, Nicholas P. Lockyer, and John C. Vickerman, A New Dynamic in Mass Spectral Imaging of Single Biological Cells. Analytical Chemistry 80 (2008) 9058-9064