ETISEQ – an algorithm for automated elution time ion sequencing of concurrently fragmented peptides for mass spectrometry-based proteomics

<p>Abstract</p> <p>Background</p> <p>Concurrent peptide fragmentation (i.e. shotgun CID, parallel CID or MS^E) has emerged as an alternative to data-dependent acquisition in generating peptide fragmentation data in LC-MS/MS proteomics experiments. Concurrent peptide fragmentation data acquisition has been shown to be advantageous over data-dependent acquisition by providing greater detection dynamic range and providing more accurate quantitative information. Nevertheless, concurrent peptide fragmentation data acquisition remains to be widely adopted due to the lack of published algorithms designed specifically to process or interpret such data acquired on any mass spectrometer.</p> <p>Results</p> <p>An algorithm called Elution Time Ion Sequencing (ETISEQ), has been developed to enable automated conversion of concurrent peptide fragmentation data acquisition data to LC-MS/MS data. ETISEQ generates MS/MS-like spectra based on the correlation of precursor and product ion elution profiles. The performance of ETISEQ is demonstrated using concurrent peptide fragmentation data from tryptic digests of standard proteins and whole influenza virus. It is shown that the number of unique peptides identified from the digests is broadly comparable between ETISEQ processed concurrent peptide fragmentation data and the data-dependent acquired LC-MS/MS data.</p> <p>Conclusion</p> <p>The ETISEQ algorithm has been designed for easy integration with existing MS/MS analysis platforms. It is anticipated that it will popularize concurrent peptide fragmentation data acquisition in proteomics laboratories.</p

The yeast Elongation Factor PAF1: Composition, Function and Interactions

An der Modulation der Chromatinstruktur und der Regulation der Transkription ist die reversible posttranslationale Modifikation der in den Nukleosomen enthaltenen Core-Histone maßgeblich beteiligt. Besonders die Funktion der co-transkriptionellen Histon-Methylierung als regulatorisches Signal für das Durchlaufen des Transkriptionszyklus konnte innerhalb der letzten Jahre immer genauer aufgeklärt werden. Der Elongationsfaktor PAF1 erweist sich durch seinen regulatorischen Einfluss auf alle drei bekannten Histon-Methyltransferasen der Hefe als entscheidender Faktor für die co-transkriptionelle Methylierung von Histon H3 und besitzt zudem eine Funktion bei der auf den Transkriptionsverlauf abgestimmten Prozessierung des 3' Endes der naszierenden mRNA. In der vorliegenden Arbeit wird die Zusammensetzung des Elongationsfaktors PAF1 sowie die Protein-Protein Interaktionen zwischen den Komponenten des Komplexes eingehend untersucht und anhand der Daten ein Modell für den Aufbau des PAF1-Komplexes vorgeschlagen. Außerdem wird der Einfluss der einzelnen Bestandteile des PAF1-Komplexes auf die Elongation und das telomere Silencing phänotypisch und funktionell charakterisiert. Zudem werden die Interaktionen des Komplexes mit dem Elongationsfaktor yFACT sowie der Casein-Kinase II untersucht und eine physikalische Wechselwirkung mit dem Bromodomainfactor 1 aufgeklärt. Der Elongationsfaktor PAF1 erweist sich dabei als pentamerer Proteinkomplex, der homogen aus den Proteinen Ctr9, Rtf1, Leo1, Paf1 und Cdc73 aufgebaut ist. Dabei zeigt sich, dass die direkte Assoziation von Paf1 und Ctr9 für die Stabilität des PAF1-Komplexes maßgeblich ist. Besonders Ctr9 wirkt aufgrund seiner seriellen Aneinanderreihung von insgesamt mindestens 10 TPR-Motiven als Bindungspartner für die übrigen Komponenten, wobei die Position der Bindungsdomänen für Paf1 und Cdc73 auf diskrete Anordnungen aus jeweils 2-3 N- bzw. C-terminal gelegenen TPR-Motiven lokalisiert werden kann. Die Voraussetzung der Paf1-Ctr9-Bindung für die Stabilität des Elongationsfaktors spiegelt sich unmittelbar in dem Befund wider, dass beide Proteine die Funktion des PAF1-Komplexes während der Elongation und auch den Einfluss auf das Telomersilencing entscheidend vermitteln. Besonders anhand der phänotypischen Untersuchung der Elongationsdefekte von Deletionsmutanten wird jedoch deutlich, dass Rtf1 und Cdc73 teilweise antagonistisch zu Paf1 wirken, was auf eine regulatorische Selbstinhibierung des Elongationsfaktors hinweisen könnte. Auch die Interaktion zwischen dem Elongationsfaktor PAF1 und yFACT sowie der Casein-Kinase II (CKII) wird durch Paf1 und Ctr9 bestimmt. Zudem konnte in vitro die CKII-abhängige Phosphorylierung von Ctr9, Rtf1, Leo1 und Paf1 gezeigt und die Lokalisation der Phosphorylierung von Ctr9 auf dessen C-Terminus begrenzt werden. Daneben konnte erstmalig die physikalische Interaktion zwischen dem Elongationsfaktor PAF1 und dem Bromo-domainfactor 1 demonstriert werden. Aufgrund der beobachteten Interaktion zwischen den Elongationsfaktoren PAF1 und yFACT sowie der Casein-Kinase II und des Bromodomainfactor 1 und anhand von Literaturdaten wird ein Modell für eine ggf. gemeinsame Beteiligung aller vier Faktoren am Capping-Kontrollpunkt vorgeschlagen und diskutiert.Modulation of chromatin structure and regulation of transcription are strongly dependent on posttranslational modifications of core histones. Especially, the importance of cotranscriptional histone methylation as a regulatory signal for progression through the transcription cycle has been realised over the past years. All three histone methyltransferases in yeast are functionally dependent on elongation factor PAF1. Additionally, the PAF1 complex has a role in coupling 3'-end processing of premature mRNA with transcription elongation and termination. This work investigates the composition of the elongation factor PAF1 in yeast and the protein-protein interactions between members within the complex. On the basis of these data a model for the assembly of PAF1 is suggested. In addition, the individual effects of its components on elongation and telomeric silencing are analysed genetically and physiologically. Furthermore, the interactions between PAF1 complex, elongation factor yFACT, and Casein Kinase II are analyzed and a physical interaction between PAF1 complex and Bromodomainfactor 1 is decribed. The data demonstrate that the elongation factor PAF1 is a homogeneously composed pentameric complex of the proteins Ctr9, Rtf1, Leo1, Paf1 and Cdc73. The association between Paf1 and Ctr9 is crucial for stability of the complex. Ctr9 acts as a binding partner for most of the other components due to its arrangement of at least 10 serial TPR motives. The location of binding domains for Paf1 and Cdc73 can be mapped to 2-3 consecutive TPR motives on the N or C-terminal end of Ctr9, respectively. The importance of Paf1 and Ctr9 for the stability of the PAF1 complex is reflected in their specific importance for transcriptional elongation and their influence on telomeric silencing. The study of individual yeast mutants suggested that both Rtf1 and Cdc73 negatively regulate the activity of Paf1. This could indicate that the function of elongation factor PAF1 is subject to negative autoregulation. The interaction between PAF1 complex, yFACT, and Casein Kinase II (CKII) are also determined by Paf1 and Ctr9. Ctr9, Rtf1, Leo1, and Paf1 were found to be phosphorylated by Casein Kinase II in vitro and the phosphorylation site(s) in Ctr9 could be mapped to its the C terminal end. Furthermore, this work demonstrates a physical interaction between PAF1 complex and Bromodomainfactor 1 (Bdf1) in yeast. On the basis of the observed interactions between the elongation factors PAF1 and yFACT as well as CKII a model for a combined participation of all four factors on the transcriptional capping checkpoint is suggested and discussed

Typing of human and animal strains of influenza virus with conserved signature peptides of matrix M1 protein by high resolution mass spectrometry

The use of high resolution mass spectrometry to detect signature peptides within proteolytic digests of the isolated matrix M1 protein, and whole virus digests, for both human and animal strains of influenza is shown to be able to rapidly and reliably type the virus. Conserved sequences for predicted tryptic peptides were identified through alignments of matrix M1 protein sequences across all human, avian and swine strains of the influenza virus. Peptides with unique masses, when compared with those from the in silico digestion of all influenza antigens and those proteins known to contaminate egg grown strains, were identified using the purpose built FluGest algorithm. Their frequency of occurrence within the matrix M1 protein across all type A and type B strains was established with the FluAlign algorithm. The subsequent detection of the signature peptides of matrix M1 protein within proteolytic digests of type A and type B human and avian strains has been demonstrated. © 2010 Elsevier B.V.Link_to_subscribed_fulltex

Rapid differentiation of seasonal and pandemic H1N1 influenza through proteotyping of viral neuraminidase with mass spectrometry

Signature peptides of the neuraminidase antigen across all common circulating human subtypes of type A and B influenza are identified through the bioinformatic alignment of translated gene sequences. The detection of these peptides within the high-resolution mass spectra of whole antigen, virus, and vaccine digests enables the strains to be rapidly and directly typed and subtyped. Importantly, unique signature peptides for pandemic (H1N1) 2009 influenza are identified and detected that enable pandemic strains to be rapidly and directly differentiated from seasonal type A (H1N1) influenza strains. The detection of these peptides can enable the origins of the neuraminidase gene to be monitored in the case of reassorted strains. © 2010 American Chemical Society.Link_to_subscribed_fulltex

Signature peptides of influenza nucleoprotein for the typing and subtyping of the virus by high resolution mass spectrometry

The use of high resolution mass spectrometry to record the accurate mass of signature peptides within proteolytic digests of the nucleoprotein antigen, and whole influenza virus, is shown to be able to rapidly type and subtype the virus. Conserved sequences for predicted tryptic peptides were identified through alignments of those for the nucleoprotein across all influenza types and subtypes. Peptides with unique theoretical masses from those generated in silico for all influenza antigen sequences, and from those proteins known to contaminate virus preparations in laboratory grown samples, were identified using a purpose built algorithm (FluGest). The frequency of occurrence of such conserved peptide signatures was assessed across all nucleoprotein sequences to subsequently type and subtype human strains of the virus. The application of the approach is illustrated for both type A H1N1 and H3N2, and type B strains of human influenza virus. © The Royal Society of Chemistry 2009.Link_to_subscribed_fulltex

Subtyping of the influenza virus by high resolution mass spectrometry

High resolution, high mass accuracy mass spectra of hemagglutinin and whole virus digests of influenza are shown to be able to be used to type and subtype the major circulating forms of the virus in humans. Conserved residues and peptide segments of the hemagglutinin antigen have been identified across type A and B strains, and for type B strains of the Yamagata 16/88 and Victoria 2/87 lineages. The theoretical masses for the protonated peptide ions for tryptic peptides of conserved sequence were subsequently shown to be unique in mass when compared to in silico generated peptides from all influenza viral protein sequences and those proteins known to contaminate virus preparations. The approach represents a more rapid and direct approach with which to type and subtype the virus that is of critical need to prepare strategies and treatments in the event of a local epidemic or global pandemic. © 2009 American Chemical Society.Link_to_subscribed_fulltex

Rapid typing and subtyping of vaccine strains of the influenza virus with high resolution mass spectrometry

The application of high-resolution mass spectrometry to type and subtype strains of the influenza virus within recent recommended vaccine formulations is described. Proteolytic digests of whole virus or separated hemagglutinin antigen generate conserved signature peptides of unique mass that can be used to characterise each component virus in a rapid and direct manner by the detection of their ions alone. The approach is demonstrated for two type A strains and one type B strain of human influenza viruses present in recommended seasonal vaccines in the northern and southern hemispheres from 2007 through 2010. © IM Publications LLP 2010.Link_to_subscribed_fulltex

Subtyping of the influenza virus by high resolution mass spectrometry

High resolution, high mass accuracy mass spectra of hemagglutinin and whole virus digests of influenza are shown to be able to be used to type and subtype the major circulating forms of the virus in humans. Conserved residues and peptide segments of the hemagglutinin antigen have been identified across type A and B strains, and for type B strains of the Yamagata 16/88 and Victoria 2/87 lineages. The theoretical masses for the protonated peptide ions for tryptic peptides of conserved sequence were subsequently shown to be unique in mass when compared to in silico generated peptides from all influenza viral protein sequences and those proteins known to contaminate virus preparations. The approach represents a more rapid and direct approach with which to type and subtype the virus that is of critical need to prepare strategies and treatments in the event of a local epidemic or global pandemic. © 2009 American Chemical Society.Link_to_subscribed_fulltex

FluTyper-an algorithm for automated typing and subtyping of the influenza virus from high resolution mass spectral data

<p>Abstract</p> <p>Background</p> <p>High resolution mass spectrometry has been employed to rapidly and accurately type and subtype influenza viruses. The detection of signature peptides with unique theoretical masses enables the unequivocal assignment of the type and subtype of a given strain. This analysis has, to date, required the manual inspection of mass spectra of whole virus and antigen digests.</p> <p>Results</p> <p>A computer algorithm, FluTyper, has been designed and implemented to achieve the automated analysis of MALDI mass spectra recorded for proteolytic digests of the whole influenza virus and antigens. FluTyper incorporates the use of established signature peptides and newly developed naïve Bayes classifiers for four common influenza antigens, hemagglutinin, neuraminidase, nucleoprotein, and matrix protein 1, to type and subtype the influenza virus based on their detection within proteolytic peptide mass maps. Theoretical and experimental testing of the classifiers demonstrates their applicability at protein coverage rates normally achievable in mass mapping experiments. The application of FluTyper to whole virus and antigen digests of a range of different strains of the influenza virus is demonstrated.</p> <p>Conclusions</p> <p>FluTyper algorithm facilitates the rapid and automated typing and subtyping of the influenza virus from mass spectral data. The newly developed naïve Bayes classifiers increase the confidence of influenza virus subtyping, especially where signature peptides are not detected. FluTyper is expected to popularize the use of mass spectrometry to characterize influenza viruses.</p