The bacterial ribosome is a complex of three strands of RNA and approximately 55 proteins. During protein synthesis, the ribosome interacts with other proteins, numbered in the hundreds, forming some stable and some transient complexes. The stoichiometries of these complexes and of partially assembled ribosomes are often unknown. We describe the development of a flexible standard for the determination of stoichiometries of ribosomal particles and complexes. A core QconCAT, an artificial protein consisting of concatenated signature peptides derived from the ribosomal proteins L2, L4, L13, S4, S7, and S8, was developed. The core QconCAT DNA construct incorporates restriction sites for the insertion of cassettes encoding signature peptides from additional proteins under study. Two cassettes encoding signature peptides from the remaining 30S and 50S ribosomal proteins were prepared, and the resulting QconCATs were expressed, digested, and analyzed by mass spectrometry. The majority of Escherichia coli ribosomal proteins are small and basic; therefore, tryptic digestion alone yields insufficient signature peptides for quantification of all of the proteins. The ribosomal QconCATs therefore rely on a dual-enzyme strategy: endoproteinase Lys-C digestion and analysis followed by trypsin digestion and further analysis. The utility of technology was demonstrated by a determination of the effect of gentamicin on the protein composition of the E. coli ribosome.

'Bottom up' proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence, enabling identification of the protein whence they derive. The majority of such studies employ collision-induced dissociation (CID) to induce fragmentation of the peptide structure giving diagnostic b-, y-, and a- ions. Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions. Such processes have the potential to adversely affect ion accounting (and thus scores from automated search algorithms) in tandem mass spectra, and in extreme cases could lead to false peptide identification. Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified. The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed. The presence of a highly basic homoarginine (or arginine) residue at the N-terminus is found to disfavor/inhibit sequence scrambling with a coincident increase in the formation of b(n-1)+H2O product ions. Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID.

The key role of the bacterial ribosome makes it an important target for antibacterial agents. Indeed, a large number of clinically useful antibiotics target this complex translational ribonucleoprotein machinery. Unfortunately, the development of resistant bacterial strains has compromised the effectiveness of most classes of antibacterial agent, including the classes that target the ribosome. Combinations of two or more drugs can be used to help overcome resistance, and in certain circumstances their action may be synergistic. In this study we have used proteomic techniques to establish the effects of gentamicin on the proteomes of aerobic and oxygen-limited Escherichia coli. Ribosomal proteins L1, L9, L10, and S2 were found to be up-regulated in both conditions, and we postulate that these are candidate drug targets for the development of synergistic combinations with gentamicin.

Glutathione reductase (Glr1) is a low abundance protein involved in defense mechanisms against reactive oxygen species. Expressed on cytosolic ribosomes, the same gene, GLR1, uses alternative start codons to generate two forms of Glr1. Translation from the first AUG codon generates the mitochondrial form incorporating a presequence necessary for import; translation from the second AUG codon yields the cytosolic counterpart. Proteomic strategies were used to analyze the N-terminal sequences and the turnover of Saccharomyces cerevisiae Glr1. The N-terminus of cytosolic Glr1 was found normally to be N-acetylserine. When a Glr1-overproducing strain was employed, unprocessed mitochondrial Glr-1 with N-terminal acetylmethionine also accumulated in the cytosol. The processed mitochondrial Glr1 was surprisingly found to have three alternative N-termini, none of them acetylated. Mitochondrial Glr1 was turned over faster than the cytosolic form by a factor of about 2, consistent with the importance of redox homeostasis in the mitochondria. These experiments also allowed us to estimate the extent of "leaky scanning" in the synthesis of Glr1. Surprisingly, the second AUG appears to be responsible for most of the cellular Glr1. This is the first report of protein turnover measurements of a low-abundance protein distributed in different compartments of a eukaryotic cell.

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a "cycle of knowledge" strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.

Production of recombinant therapeutic proteins, especially monoclonal antibodies (Mab), in myeloma cell lines represents a significant segment of the pharmaceutical market, and therefore striving for increased productivity of these lines represents a major investment of resources. The elucidation of biologically important markers for the adaptation of NS0 myeloma cell line to protein-free medium and the recombinant protein production are a major emphasis of our research. These markers could potentially be used in a variety of ways to improve culture conditions, including active approaches to agonize/antagonize important pathways within a medium formulation or diagnostic approaches indicative of improved conditions during the culture. In this work, we used two-dimensional electrophoresis/mass spectrometry and the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 myeloma cell line. Several proteins with differential expression profile were characterized and quantified. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation by proteomic analysis. The same results were obtained using flux balance analysis in a murine metabolic network with selected medium conditions.

A typical ‘bottom up’ proteomic workflow uses tandem mass spectrometric data to infer product ion sequence and hence identity of the protein from which they derive. Such analysis is typically performed following proteolysis with the endoproteases trypsin or Lys-C; peptides produced therefore terminate in the basic residues arginine or lysine. Removal of these C-terminal basic residues using the exopeptidase, carboxypeptidase B, generates peptides whose analysis by tandem MS yields evidence of substantially different fragmentation properties. The decompositions of peptide ions both prior to and following treatment with carboxypeptidase B have been examined using collision-induced dissociation and electron transfer dissociation. Changes in properties following secondary enzyme treatment are attributed primarily to removal of a strongly basic site, with a consequent effect both on the propensity to retain charge and the stability of the fragment ions. The data suggest a complementary value in proteome analyses for MS/MS of tryptic/Lys-C peptides with and without subsequent carboxypeptidase B treatment.

The development of ion mobility (IM) MS instruments has the capability to provide an added dimension to peptide analysis pipelines in proteomics, but, as yet, there are few software tools available for analysing such data. IM can be used to provide additional separation of parent ions or product ions following fragmentation. In this work, we have created a set of software tools that are capable of converting three dimensional IM data generated from analysis of fragment ions into a variety of formats used in proteomics. We demonstrate that IM can be used to calculate the charge state of a fragment ion, demonstrating the potential to improve peptide identification by excluding non‐informative ions from a database search. We also provide preliminary evidence of structural differences between b and y ions for certain peptide sequences but not others. All software tools and data sets are made available in the public domain at http://code.google.com/p/ion-mobility-ms-tools/.

Ion mobility-mass spectrometry (IM-MS) is a useful technique for determining information about analyte ion conformation in addition to mass/charge ratio. The physical principles that govern the mobility of an ion through a gas in the presence of a uniform electric field are well understood, enabling rotationally averaged collision cross sections (Ω) to be directly calculated from measured drift times under well-defined experimental conditions. However, such "first principle" calculations are not straightforward for Traveling Wave (T-Wave) mobility separations due to the range of factors that influence ion motion through the mobility cell. If collision cross section information is required from T-Wave mobility separations, then calibration of the instruments using known standards is essential for each set of experimental conditions. To facilitate such calibration, we have designed and generated an artificial protein based on the QconCAT technology, QCAL-IM, which upon proteolysis can be used as a universal ion mobility calibration standard. This single unique standard enables empirical calculation of peptide ion collision cross sections from the drift time on a T-Wave mobility instrument.

Reversible phosphorylation regulates the majority of intracellular networking and pathways. The study of this widely explored post-translational modification is usually challenged by low stoichiometric levels of modification. Many approaches have been developed to overcome this problem and to achieve rigorous characterization of protein phosphorylation. We describe a method for enhanced detection of low-abundance protein phosphorylation that uses selective introduction of (18)O label into phosphorylation sites with H(2)(18)O and mass spectrometric detection. The method was applied to introduce (18)O label into bacterially expressed Aurora A kinase phosphorylation sites and resulted in the representation of phosphorylated peptides as doublets or triplets according to the number of phosphate groups. A total of 28 phosphopeptides were observed by this method.

Rapid Communications in Mass SpectrometryVolume 26, Issue 2 p. 205-206 Letter to the Editor Proposal for a common nomenclature for peptide fragment ions generated following sequence scrambling during collision-induced dissociation Ross Chawner, Ross Chawner Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester, M1 7DN UKSearch for more papers by this authorSimon J. Gaskell, Simon J. Gaskell Queen Mary University of London, London, E1 4NS UKSearch for more papers by this authorClaire E. Eyers, Corresponding Author Claire E. Eyers Claire.Eyers@manchester.ac.uk Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester, M1 7DN UK C. Eyers, Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester M1 7DN, UK. E-mail: Claire.Eyers@manchester.ac.ukSearch for more papers by this author Ross Chawner, Ross Chawner Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester, M1 7DN UKSearch for more papers by this authorSimon J. Gaskell, Simon J. Gaskell Queen Mary University of London, London, E1 4NS UKSearch for more papers by this authorClaire E. Eyers, Corresponding Author Claire E. Eyers Claire.Eyers@manchester.ac.uk Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester, M1 7DN UK C. Eyers, Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester M1 7DN, UK. E-mail: Claire.Eyers@manchester.ac.ukSearch for more papers by this author First published: 05 December 2011 https://doi.org/10.1002/rcm.5294Citations: 8Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume26, Issue230 January 2012Pages 205-206 RelatedInformation

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.

The dissociation of protein ions (5-30  kDa) as a function of charge state has been explored in order to suggest the optimal charge state range for top-down sequencing. Proteins were generated under denaturing conditions and their charge states were modified via ion/ion proton transfer reactions prior to dissociation. Electron transfer dissociation (ETD) data suggested optimal sequence coverage for charge states in the m/z range from 700 to 950 while limited sequence coverage was noted when the precursor m/z was above 1000. Sequence coverage from ETD data was found to be dependent on protein size, with smaller proteins having better sequence coverage. An observed depletion in sequence-related information was mainly attributed to limited instrument (ion trap) performance (m/z range and resolution). For a combined ETD/collision-induced dissociation (CID) approach it is difficult to propose an optimal m/z range since good sequence coverage for CID is at intermediate charge states and the optimal m/z range increases with protein size. When only one charge state can be analysed in a combined ETD/CID approach, a range around 950  m/z is suggested as a starting point. Alternatively, two charge states should be explored, each optimal for either ETD or CID. Overall, these suggestions should be useful to achieve enhanced characterisation of smaller proteins/large protein fragments (generated from denaturing solutions) in minimal analysis times.

In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy: light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.

We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)-5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine.

The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LCMS, using ultra high performance liquid chromatographycoupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/ cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods. Molecular & Cellular Proteomics 10: 10.1074/mcp. M111.007633, 1-15, 2011.

Mass spectrometry has emerged as a powerful tool for the analysis of all lipids. Lipidomic analysis of biological systems using various approaches is now possible with a quantitative measurement of hundreds of lipid molecular species. Although availability of reference and internal standards lags behind the field, approaches using stable isotope-labeled derivative tagging permit precise determination of specific phospholipids in an experimental series. The use of reactivity of ozone has enabled assessment of double bond positions in fatty acyl groups even when species remain in complex lipid mixtures. Rapid scanning tandem mass spectrometers are capable of quantitative analysis of hundreds of targeted lipids at high sensitivity in a single on-line chromatographic separation. Imaging mass spectrometry of lipids in tissues has opened new insights into the distribution of lipid molecular species with promising application to study pathophysiological events and diseases.