An essential role for the Michael Barber Centre

The development of proteomics has been based very heavily on the suite of analytical techniques encompassed by mass spectrometry and associated methods. It is therefore appropriate that the work of the Michael Barber Centre for Mass Spectrometry (MBCMS, named for the inventor of, inter alia, the fast atom bombardment ionisation method) should now be largely driven by the needs of proteomics research and the broader field of systems biology.

This scientific context establishes rather clearly the analytical priorities for the MBCMS. In the first instance there is a need in the proteomics field for analytical methods of enhanced sensitivity and selectivity. Developments in mass spectrometry instrumentation in recent years have certainly led to marked improvements in sensitivity, but our current capabilities (typically analysis at the femtomole level) are still modest by comparison with the sensitivities of biological recognition processes and mean that the objective of ‘ single-cell proteomics’ is an extreme challenge. The analytical parameters of sensitivity and selectivity are distinct but rather closely related. Specifically, can we develop analytical strategies that achieve just the requisite degree of selectivity while maximising sensitivity? And what defines the selectivity required? These qualitative analytical imperatives are supplemented by a second priority to improve quantitative procedures, where the analytical strategies may be informed by the long history of the use of mass spectrometry for the quantitative determination of small molecules.

Qualitative analytical requirements

The proteomics field is burdened with an abundance of acronyms, sometimes disguising a lack of clarity in addressing key analytical questions. Key amongst the latter is the issue of what constitutes the protein ‘identification’ that is often claimed in proteomics experiments. A standard proteomics workflow commonly includes proteolytic digestion of proteins, with partial separation either at the protein or peptide level. The digest is then analysed by conventional mass spectrometry, yielding a (partial) list of proteolytic peptide masses and by tandem mass spectrometry, yielding (partial) sequence information. These data are then used in database searches where the peptide masses and sequence data (often in uninterpreted form) constitute the search terms. The confidence in recognition of the protein from which the analytical data are derived is clearly dependent on the extent of matched sequence, and there is much debate as to what constitutes an acceptable protein match. The truth, however, is that we almost never obtain data derived from a full sequence, so that the process is not so much one of ‘identification’ as the establishment of sufficient congruity of sequence between the biological isolate and a database sequence that we can infer a common genetic origin. That being the case, the analytical imperative becomes one of maximising sensitivity in the achievement of the minimum extent of analytical data to draw the common origin conclusion – an outcome better described (if shorthand is needed) as protein ‘recognition’, rather than ‘identification’.

Such considerations should prompt questions concerning the optimisation of the mass spectrometric methods employed. In the MBCMS we are particularly concerned with the improvement of the tandem mass spectrometry experiment, either by adjustment of the technique itself or by modification of the analyte (peptide) structure prior to mass spectrometric analysis in order to enhance the yield of diagnostic information. Simple examples of the latter include the use of guanidination of lysine side-chains to achieve increased detectability during matrix-assisted laser desorption mass spectrometry and to confer gas-phase fragmentation properties akin to those observed with arginine-containing analogues1,2.

Equally straightforward chemistry (though its application at the femtomole level required considerable development) is involved in the use of phenylthiocarbamoyl derivatives to induce specific fragmentation properties of protonated peptide ions following collisional activation in a tandem mass spectrometry experiment. This is Edman chemistry in the gas phase – low energy collisional activation of the derivatised peptides, when analysed as the doubly protonated species commonly generated by electrospray, leads to fragmentation predominantly at the N-terminal peptide bond3. The resulting product ion spectrum yields only the identity of the N-terminal amino acid residue but this information is obtained with high sensitivity as a result of the concentration of product ion signal solely in the products of a single cleavage process. The modest yield of structural information may nevertheless be sufficient in certain instances, such as the recognition of proteins in simple organisms with sequenced genomes4. This adjustment of the sensitivity/selectivity balance provides a simple example of the use of analyte modification to meet specific analytical requirements. In particular, we are exploiting this approach to achieve highly selective targeted analysis of specific peptide sub-groups (Riba-Garcia et al., unpublished work).

Optimisation of the technique itself, rather than adjustment of analyte structure, represents a second general approach to the improvement of mass spectrometric methods for proteome analysis. The increasing reliance on tandem mass spectrometry should prompt questions as to whether our current methods are optimal. The uncomfortable truth is that tandem mass spectrometry as generally applied is a crude experiment – we isolate a specific precursor ion, heat it (generally by collisional activation, but sometimes using a laser), and hope that the resulting fragmentation is efficient and informative. Recent developments encourage some optimism that more sophisticated approaches may yield substantial dividends. The recently introduced method of electron transfer dissociation5 involves reaction of multiply protonated analyte ions with reagent anions, resulting in electron transfer and the promotion of radical-induced ion cleavage. When applied to peptide ions, the observed fragmentations are quite distinct from those promoted by collisional activation. In a recent study in collaboration with colleagues at Thermo Electron6, the combined application of electron transfer dissociation and collision induced dissociation enabled the recognition of a much larger number of proteins in the flagella of Trypanosoma bruceii than was possible using conventional collision induced dissociation alone7. This example illustrates a further keypoint that is central to the work of the MBC, namely that the most significant analytical development work is conducted explicitly within the context of the demands identified by the biological researchers – in this case Professor Keith Gull (University of Oxford), Dr Paul McKean (University of Lancaster) and their colleagues.

Quantitative analytical requirements

The need to supplement a qualitative description of a proteome with quantitative data is increasingly widely recognised. There is, however, insufficient appreciation of the breadth of quantitative questions to be answered. Too commonly, ‘quantitative proteomics’ is considered to refer only to the study of changes in protein expression in comparing two (or more) related samples, such as cell cultures before and after xenobiotic treatment. This is certainly an important topic (and is considered in detail below), but the full definition of a proteome (though this is not always essential) also requires a description of individual protein concentrations in absolute terms, such as copy number per cell. Furthermore, a particular absolute protein concentration may be achieved through different combinations of rates of synthesis and degradation, so that estimates of protein turnover are also essential for complete definition of a proteome. Such a comprehensive treatment of quantitative issues is increasingly important as proteomics finds its place in the broader context of systems biology; certainly, such ideas are integral to the thinking behind the Manchester Centre for Integrative Systems Biology, led by Prof. Douglas Kell and of which the Michael Barber Centre is a member.

Rigorous approaches to relative quantification in proteomics are based on principles of differential labelling, which may be fluorescent (as in the differential gel electrophoresis, or DiGE, method8) or isotopic. The combination of stable isotope labelling with mass spectrometric detection is particularly powerful and has been widely exploited in quantitative proteomics. When the objective is relative quantification, differential isotope labelling may be achieved using one of three general strategies. When circumstances permit, the ideal approach is arguably the incorporation of isotopic label at the metabolic level, for example by growing the organism of interest in an isotopically distinct medium. An example of this approach is the SILAC method (stable isotope labelling with amino acids in culture9), in which the growth medium is supplemented by one or more isotopically labelled amino acids. Where this is difficult or impossible (as is generally the case in studying higher organisms), the isotopic distinction between the samples to be compared may be achieved by labelling of protein fractions, as in the ICAT approach (isotope coded affinity tagging10). Finally, there may be analytical benefit to postponement of differential isotope labelling until protein samples have been hydrolysed; such peptide labelling is employed, for example, in the ITRAQ approach (isobaric tagging for relative and absolute quantification11). Our own work in this area has emphasised the value of employing labelling approaches in which the derivatisation technique confers multiple advantages of improved mass spectrometric detection, differential labelling, and enhanced ion fragmentation properties2. The role of tandem mass spectrometry may be key: the enhanced analytical selectivity achieved in this way can be critical and the detection of multiple fragment ions can yield useful replication of isotope ratio determination.

The extension of relative quantification in proteomics to absolute quantification is conceptually trivial – both share the practice of using proteolytic peptides as analytical surrogates for the proteins from which they are derived and absolute quantification is, of course, only relative quantification where one of the values is known. The real challenge for absolute quantification is the generation of sufficient supplies of large numbers of authentic peptide standards in known amounts. In a collaborative programme led by Prof. Rob Beynon of the University of Liverpool, we have developed the QconCAT approach12 (www.entelechon.com), in which artificial proteins are designed, corresponding to the concatenation of multiple tryptic peptides, each serving as a signature peptide for a protein of interest. Introduction and expression of the corresponding DNA vector in E. coli enables the harvesting of the artificial protein, which then serves as internal standard for the absolute quantification of multiple proteins (for example, using the ITRAQ approach). Growth of the bacterium in an isotopically labelled medium (incorporating, for example, deuterium-labelled arginine and lysine) yields a labelled protein standard.

The quantitative definition of a proteome, even in absolute terms, is incomplete if no consideration is given to the balance of protein synthesis and degradation that yields the net concentrations of individual proteins. Again in collaboration with the Beynon group, we have worked for several years on the development and application of stable isotope labelling approaches to the study of protein turnover, both in yeast13,14 and in higher organisms15. Challenges in protein dynamics remain, notably the determination of the kinetics of post-translational modification.

In summary, the approach taken to proteomics research at the Michael Barber Centre includes an acknowledgement of the essential role of both qualitative and quantitative analyses. It is critical also to recognise that the most challenging biological questions can be addressed only by developing and exploiting the full capabilities of the enabling physical chemical techniques. Such a philosophy is no more than an example of the exploitation of the life sciences/physical sciences interface, a general approach that underpins the Manchester Interdisciplinary Biocentre, in which the Michael Barber Centre is now placed.

About the Michael Barber Centre

The Centre, directed by Professor Simon Gaskell, was established in 1990 under the directorship of the late Professor Michael Barber FRS as a joint venture between UMIST, other universities and industry. It was renamed the Michael Barber Centre for Mass Spectrometry in 1994, in recognition of the distinguished contributions to the science of mass spectrometry made by Professor Barber. UMIST and The Victoria University of Manchester joined forces in 2004 to create The University of Manchester.

The interests and expertise of the Centre are in the development of mass spectrometry and related analytical techniques and their application to problems of biological importance. In particular, the Centre is concerned with the understanding of the gas-phase ion chemistry of biomolecules (notably peptides and proteins) and the exploitation of that understanding in enhanced analytical approaches. Much of his current effort is devoted to proteomics (the definition of the protein complement of cells and organisms), with particular reference to quantitative determinations.

The Centre is the lead partner in a newly funded consortium (funded by the EPSRC) that aims to develop new analytical approaches to the characterisation and quantification of the proteome. We are major participants in the Manchester Centre for Integrative Systems Biology. In multiple other programmes, we work with multiple collaborators in many disciplines (biology, informatics, clinical science, physics) and from many partner institutions (e.g. Liverpool, Oxford, Cambridge, Lancaster). The Centre has state-of-the-art instrumentation, including the following: ion cyclotron resonance, Q-ToF, quadrupole ion trap, MALDI-ToF, ESI-ToF, tandem quadrupole.

References

1. Brancia FL, Oliver SG, Gaskell SJ. Improved Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Tryptic Hydrolysates of Proteins Following Guanidination of Lysine-Containing Peptides. Rapid Commun. Mass Spectrom. 2000; 14 (21): 2070-2073.
2. Warwood S, Mohammed S, Cristea IM, Evans C et al. Guanidination Chemistry for Qualitative and Quantitative Proteomics. Rapid Comm. Mass Spectrom. 2006; in press.
3. Summerfield SG, Bolgar MS, Gaskell SJ. Promotion and Stabilization of B(1) Ions in Peptide Phenylthiocarbamoyl Derivatives: Analogies With Condensed-Phase Chemistry. J Mass Spectrom. 1997; 32 (2): 225-231.
4. Sidhu KS, Sangvanich P, Brancia FL, Sullivan AG et al. Bioinformatic Assessment of Mass Spectrometric Chemical Derivatisation Techniques for Proteome Database Searching. Proteomics 2001; 1: 1368-1377.
5. Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J et al. Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry. Proceedings of the National Academy of Sciences of the United States of America. JID – 7505876 2004; 101 (26): 9528-9533.
6. Enhanced Protein Identification Using Electron Transfer Dissociation and Collision Induced Dissociation: A Study of the Trypanosoma brucei Flagellar Proteome.: 2006.
7. Broadhead R, Dawe HR, Farr H, Griffiths S et al. Flagellar Motility Is Required for the Viability of the Bloodstream Trypanosome. Nature. JID – 0410462 2006; 440 (7081): 224-227.
8. Tonge R, Shaw J, Middleton B, Rowlinson R et al. Validation and Development of Fluorescence Two-Dimensional Differential Gel Electrophoresis Proteomics Technology. Proteomics 2001; 1: 377-396.
9. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB et al. Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, As a Simple and Accurate Approach to Expression Proteomics. Molecular & Cellular Proteomics : MCP. JID – 101125647 2002; 1 (5): 376-386.
10. Gygi SP, Rist B, Gerber SA, Turecek F et al. Quantitative Analysis of Complex Protein Mixtures Using Isotope-Coded Affinity Tags. Nat. Biotechnol. 1999; 17: 994-999.
11. Pappin DJC, Barlet-Jones M. Methods, mixtures, kits and compositions pertaining to analyte determination. patent WO 2004/070352. 2004 2004.
12. Beynon RJ, Doherty MK, Pratt JM, Gaskell SJ. Multiplexed Absolute Quantification in Proteomics Using Artificial QCAT Proteins of Concatenated Signature Peptides. Nature Methods. JID – 101215604 2005; 2 (8): 587-589.
13. Pratt JM, Petty J, Riba-Garcia I, Robertson DH et al. Dynamics of Protein Turnover, a Missing Dimension in Proteomics. Mol. Cell Proteomics 2002; 1: 579-591.
14. Pratt JM, Robertson DH, Gaskell SJ, Riba-Garcia I et al. Stable Isotope Labelling in Vivo As an Aid to Protein Identification in Peptide Mass Fingerprinting. Proteomics 2002; 2: 157-163.
15. Doherty MK, McLean L, Hayter JR, Pratt JM et al. The Proteome of Chicken Skeletal Muscle: Changes in Soluble Protein Expression During Growth in a Layer Strain. Proteomics 2004; 4: 2082-2093.

About the author – Simon J Gaskell

Prof. Gaskell has directed the Michael Barber Centre since 1993, having moved to Manchester (then the University of Manchester Institute of Science and Technology, before a merger in 2004 with the Victoria University of Manchester) from Baylor College of Medicine, Houston, USA. For many years he has focused on the development and application of mass spectrometry to the solution of problems in biological research.

Dr Riba obtained her first degree at the University of Salamanca, Spain, before studying for her PhD in the Michael Barber Centre. Following a period in the pharmaceutical industry, she returned to Manchester, where she is now a Research Fellow. Her expertise lies in the qualitative and quantitative analysis of compounds of biological importance (both endogenous and xenobiotic) using mass spectrometry and ancillary techniques.