Hybrid-FT-Massenspektrometer
Zusammenfassung der Projektergebnisse
1) High accuracy MS in refinement of genome sequencing data Mass spectrometry-based proteomics is increasingly used in refinement of genome annotation. The typical “proteogenomic” workflows rely on mapping of peptide MS/MS spectra onto databases derived by six-frame translation of the genome sequence; these databases contain a large proportion of spurious protein sequences which make the statistical confidence of the resulting peptide spectrum matches (PSMs) difficult to assess. Influence of large and asymmetric search space has so far been subject of intense discussion, but a precise assessment of the main parameters of a proteogenomic dataset – sensitivity, specificity, accuracy, actual false discovery rate (FDR) and overall genome coverage by detected peptide sequences – is still missing in the literature. In this project we performed a comprehensive analysis of the Escherichia coli proteome, collecting ca. 2M MS/MS spectra on LTQ-Orbitrap velos MS. We achieved complete proteome coverage and mapped the corresponding MS/MS spectra onto a six-frame translation of the E. coli genome. Since E. coli is one of the most investigated model organisms, we assumed complete annotation of its genome; this simple assumption enabled us to regard all six frame-specific (novel) PSMs as false positive identifications and permits us to assess the main parameters of a typical bacterial proteogenomic dataset. We showed that the posterior error probability distribution of six frame-specific (novel) hits is almost identical to that of reversed (decoy) hits, pointing to substantial underestimation of FDR even in simple proteogenomic experiments obtained by high accuracy MS. The use of a small and well annotated bacterial genome also enabled us to address genome coverage achieved in state-of-the-art bacterial proteomics: identified peptide sequences led to detection of all E. coli proteins expected to be expressed at the point of analysis, but covered only 27.5% of the total genome sequence. Combined, these results provide a precise analysis of the major outcomes of a simple (bacterial) proteogenomic experiment and point to the necessity for further technological and bioinformatic improvements in proteogenomic strategies. 2) Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast) To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in S. pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis. 3) Central Project of the SFB 766: Identification and quantitation of bacterial membrane proteome (DFG, SFB766/Z2b) As a Central Project of the SFB766 “The Bacterial Cell Envelope: Structure, Function and Infection Interface”, PCT offers state-of-the-art methodology for isolation, identification and quantification of bacterial membrane proteins to all members of the SFB. In the second funding period (2011-2015) the PCT has provided qualitative and quantitative analyses of proteins and protein modifications such as phosphorylation or lysine acetylation (to A02 Wohlleben/Muth, A07 Wolz/Weidenmaier, A11 Forchhammer/Maldener, B01 Schütz, B07 Gust, B11 Rapaport); quantitative analyses of sub-proteomes, such as isolated protein fractions or immunoprecipitates (to A08 Peschel, A11 Forchhammer/Maldener, B07 Gust, B11 Rapaport, B14 Wagner) and analyses of whole proteomes (A11 Forchhammer/Maldener). In these collaborations we performed more than 1000 LC-MS measurements, about half of them on the LTQ Orbitrap Velos instrument. 4) Core Facility The Proteome Center Tuebingen serves as a core facility of the University of Tuebingen and has separate Core Facility Agreements with the University Clinic Tuebingen and the Max-Planck- Institute for Developmental Biology, Tuebingen. The LTQ Orbitrap Velos funded by the DFG is one of only two mass spectrometers operated by the PCT and was therefore heavily involved in core facility projects. From installation of the instrument in May 2011, the PCT processed more than 300 core facility projects.
Projektbezogene Publikationen (Auswahl)
- 2012. Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA-processing. Cell 151(4):859-70
Manavella, P.A., Hagmann, J., Ott, F., Laubinger, S., Franz-Wachtel, M., Macek, B., Weigel, D.
- 2013. Deep coverage of the Escherichia coli proteome enables the assessment of database search strategies in bacterial proteogenomics experiments. Mol Cell Proteomics 12(11):3420-30
Krug, K., Carpy, A., Behrends, G., Matic, K., Soares, N.C., Macek, B.
- 2013. Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium. J Prot Res 12(6):2611-21
Soares, N.C., Spät, P., Krug, K., Macek, B.
- 2014. Absolute proteome and phosphoproteome dynamics during the cell cycle of fission yeast. Mol Cell Proteomics 13(8):1925-36
Carpy, A., Krug, K., Graf, S., Koch, A., Popic, S., Hauf, S., Macek, B.
(Siehe online unter https://doi.org/10.1074/mcp.M113.035824) - 2014. MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumaniae. Nature 516(7530):259-62
Fleurie, A., Lesterlin, C., Manuse, S., Zhao, C., Cluzel, C., Lavergne, J-P., Franz-Wachtel, M., Macek, B., Combet, C., Kuru, E., VanNieuwenhze, M.S., Brun, Y.V., Sherratt, D., Grangeasse, C.
(Siehe online unter https://doi.org/10.1038/nature13966)
