"REFINING TANDEM MASS SPECTROMETRY FOR THE ANALYSIS OF PEPTIDES AND"
REFINING TANDEM MASS SPECTROMETRY FOR THE ANALYSIS OF PEPTIDES AND PROTEINS Simon J Gaskell Michael Barber Centre for Mass Spectrometry and Manchester Centre for Integrative Systems Biology, University of Manchester, Manchester, UK - Simon. Gaskell@manchester.ac.uk Tandem mass spectrometry is routinely used in proteome analyses to provide sequence information (commonly incomplete) for proteolytic peptides. The data so obtained provide additional search terms by which the proteins from which the peptides are derived may be recognised using database searching. The elegance of the overall approach belies the lack of sophistication of one aspect of the tandem mass spectrometry experiment itself (as most commonly employed) namely the collisionally activated dissociation (CAD) of selected precursor ions. Thus, the production of fragment ions from which structural information may be derived is promoted by collision of precursors with neutral gas atoms or molecules, resulting in conversion of kinetic energy to internal energy. In general, there is a modest degree of control over the extent of fragmentation but almost no control over the direction of fragmentation. Fortunately, the unimolecular decomposition chemistry of protonated peptides is commonly informative, though much remains to be learnt concerning mechanistic details. Nevertheless, there are significant limitations to CAD, notably the difficulty in characterisation of post-translationally modified species, such as phosphopeptides. In addition, the facts that CAD corresponds essentially to an ion heating method, and that excess internal energy has ample time for equilibration through all degrees of freedom in the time frame of the decomposition experiment, mean that the fragmentation efficiency declines quite steeply with increasing molecular weight of the peptide analyte. Here we discuss approaches to enhancement of the tandem MS experiment, with particular reference to its use in both structure elucidation and trace analysis. When the objective is the improvement of detectability of targeted components, it is helpful to achieve a concentration of fragment ion signal in one or a few product ions. This may be achieved by selective peptide derivatisation (in the condensed phase prior to MS analysis) in order to direct fragmentation along energetically preferred pathways. Conversion of the N-terminal amine group to a nitrophenyl thiocarbamoyl derivative, for example, promotes the equivalent of Edman cleavage in the gas phase of the N-terminal amide bond, with production of yn-12+ ions. At low collision energies, these are commonly the only fragments observed, making them ideal candidates for detection during trace analyses using selected reaction monitoring. In other instances, the objective is the enhancement of the extent of diagnostically informative data. The recently developed and related techniques of electron capture dissociation and electron transfer dissociation (ETD) show considerable promise in this respect. The latter is achieved by ion/ion reaction between peptide precursor and a reagent anion; this represents the first substantial application of bimolecular gas-phase chemistry to biomolecule analysis. Here we describe the application of ETD in proteome analyses and report the enhanced information obtained and the complementarity with CAD analyses. Acknowledgements: This work has benefited from multiple inputs from many members of the Michael Barber Centre (notably Drs Sarah Hart and Isabel Riba-Garcia), from collaborations with the research groups of Prof. Keith Gull (University of Oxford), Dr Paul McKean (University of Lancaster), and from the assistance of colleagues at Thermo Electron and Bruker. The work is supported by the UK EPSRC and BBSRC.