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ABSTRACT: Databases which capture proteomic data for subsequent interrogation can be extremely useful for our understanding of peptide ion behaviour in the mass spectrometer, leading to novel hypotheses and mechanistic understanding of the underlying mechanisms determining peptide fragmentation behaviour. These, in turn, can be used to improve database searching algorithms for use in automated and unbiased interpretation of peptide product ion spectra. Here, we examine a previously published dataset using our established methods, in order to discover differences in the observation of product ions of different types, following ion activation and unimolecular dissociation either by collisional dissociation or the ion/ion reaction, electron transfer dissociation. Using a target-decoy database searching strategy, a large data set of precursor ions, were confidently predicted as peptide sequence matches (PSMs) at either a 1% or 5% peptide false discovery rate, as reported in our previous study. Using these high quality PSMs, we have conducted a more detailed and novel analysis of the global trends in observed product ions present/absent in these spectra, examining both CID and ETD data. We uncovered underlying trends for an increased propensity for the observation of higher members of the ion series in ETD product ion spectra in comparison to their CID counterparts. Such data-mining efforts will prove useful in the generation of new database searching algorithms which are well suited to the analysis of ETD product ion spectra.
Methods in molecular biology (Clifton, N.J.) 01/2011; 696:327-37.
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Sarah R Hart
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ABSTRACT: Analysis of intact proteins by tandem mass spectrometry has mostly been confined to high-end mass spectrometry platforms. This protocol describes the application of routine HPLC to separate proteins, MALDI-ToF mass spectrometry to interrogate intact protein species and electron transfer dissociation/proton transfer reaction within a quadrupole ion trap to perform tandem mass spectrometry.
Methods in molecular biology (Clifton, N.J.) 01/2010; 658:339-53.
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ABSTRACT: Tandem mass spectrometric data from peptides are routinely used in an unsupervised manner to infer product ion sequence and hence the identity of their parent protein. However, significant variability in relative signal intensity of product ions within peptide tandem mass spectra is commonly observed. Furthermore, instrument-specific patterns of fragmentation are observed, even where a common mechanism of ion heating is responsible for generation of the product ions. This information is currently not fully exploited within database searching strategies; this motivated the present study to examine a large dataset of tandem mass spectra derived from multiple instrumental platforms. Here, we report marked global differences in the product ion spectra of protonated tryptic peptides generated from two of the most common proteomic platforms, namely tandem quadrupole-time-of-flight and quadrupole ion trap instruments. Specifically, quadrupole-time-of-flight tandem mass spectra show a significant under-representation of N-terminal b-type fragments in comparison to quadrupole ion trap product ion spectra. Energy-resolved mass spectrometry experiments conducted upon test tryptic peptides clarify this disparity; b-type ions are significantly less stable than their y-type N-terminal counterparts, which contain strongly basic residues. Secondary fragmentation processes which occur within the tandem quadrupole-time-of-flight device account for the observed differences, whereas this secondary product ion generation does not occur to a significant extent from resonant excitation performed within the quadrupole ion trap. We suggest that incorporation of this stability information in database searching strategies has the potential to significantly improve the veracity of peptide ion identifications as made by conventional database searching strategies.
Rapid Communications in Mass Spectrometry 04/2009; 23(10):1508-14. · 2.79 Impact Factor
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ABSTRACT: The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD- or ETD-based tandem mass spectrometry alone.
Journal of the American Society for Mass Spectrometry 10/2008; 20(2):167-75. · 4.00 Impact Factor
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SEB experimental biology series. 02/2008; 61:37-64.
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Juan I Castrillo,
Leo A Zeef,
David C Hoyle,
Nianshu Zhang,
Andrew Hayes,
David C J Gardner,
Michael J Cornell,
June Petty,
Luke Hakes,
Leanne Wardleworth, [......],
Svenja S Hester,
Tom P J Dunkley, Sarah R Hart,
Neil Swainston,
Peter Li,
Simon J Gaskell,
Norman W Paton,
Kathryn S Lilley,
Douglas B Kell,
Stephen G Oliver
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ABSTRACT: Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking.
Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth.
This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell.
Journal of Biology 02/2007; 6(2):4.
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ABSTRACT: Proteomics continues to play a critical role in post-genomic science as continued advances in mass spectrometry and analytical chemistry support the separation and identification of increasing numbers of peptides and proteins from their characteristic mass spectra. In order to facilitate the sharing of this data, various standard formats have been, and continue to be, developed. Still not fully mature however, these are not yet able to cope with the increasing number of quantitative proteomic technologies that are being developed.
We propose an extension to the PRIDE and mzData XML schema to accommodate the concept of multiple samples per experiment, and in addition, capture the intensities of the iTRAQ reporter ions in the entry. A simple Java-client has been developed to capture and convert the raw data from common spectral file formats, which also uses a third-party open source tool for the generation of iTRAQ reported intensities from Mascot output, into a valid PRIDE XML entry.
We describe an extension to the PRIDE and mzData schemas to enable the capture of quantitative data. Currently this is limited to iTRAQ data but is readily extensible for other quantitative proteomic technologies. Furthermore, a software tool has been developed which enables conversion from various mass spectrum file formats and corresponding Mascot peptide identifications to PRIDE formatted XML. The tool represents a simple approach to preparing quantitative and qualitative data for submission to repositories such as PRIDE, which is necessary to facilitate data deposition and sharing in public domain database. The software is freely available from http://www.mcisb.org/software/PrideWizard.
Proteome Science 02/2007; 5:4. · 2.33 Impact Factor
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Richard Broadhead,
Helen R Dawe,
Helen Farr,
Samantha Griffiths, Sarah R Hart,
Neil Portman,
Michael K Shaw,
Michael L Ginger,
Simon J Gaskell,
Paul G McKean,
Keith Gull
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ABSTRACT: The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.
Nature 04/2006; 440(7081):224-7. · 36.28 Impact Factor
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ABSTRACT: The coupling of high-performance liquid chromatography (HPLC) to mass spectrometry (MS) has been routinely used for a number of years for the analysis of a wide variety of different compounds. In typical proteomic analyses, where enzymatic digestion is used to generate proteolytic peptides, the limited amount of sample restricts the utility of conventional HPLC methods for MS detection. As reduced column diameters and nanolitre per minute flow rates have become increasingly standard, the application of HPLC to the analysis of low-volume, low-abundance samples has now become readily achievable. A number of novel chromatographic methods have increased the utility of such approaches for proteome-wide analyses. However, there remain in proteomic analyses some important challenges, which are being addressed by state-of-the-art methodologies. This article reviews a number of pertinent considerations and technological advances in proteomic analyses using HPLC–tandem MS (MS2).
TrAC Trends in Analytical Chemistry.
