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Proteomanalytik

Die Arbeitsgruppe Proteomanalytik verbindet modernste Proteom-Wissenschaft mit der Pflanzenforschung. Es ist unser Forschungsgebiet, die dynamische Umstrukturierung des Proteoms, d. h. die Proteinabundanz, sub-zelluläre Lokalisierung, posttranslationale Modifikation und Proteininteraktion in diversen biologischen Szenarien quantitativ zu erfassen. Zu diesem Zweck bedient sich die Arbeitsgruppe der ultra-performance Flüssigkeitschromatographie gekoppelt mit der hochauflösenden Massenspektrometrie.

Abb.1 und 2. Moderne Massenspektrometrie zur Peptid- und Proteinmessung.
Abb.1 und 2. Moderne Massenspektrometrie zur Peptid- und Proteinmessung.

Kürzlich haben wir eine pflanzenspezifische Deep-Proteomics Strategie etabliert und optimiert. Diese macht es möglich zwischen 6,000 und 9,000 Proteine aus einer Pflanzengewebeprobe zu quantifizieren. Sie werden zusammen mit Metabolomics und gerichteter Proteomanalyse angewandt um eine Rolle des Pflanzenhormones Auxin/IAA in der Arabidopsis thaliana Immunantwort näher zu beleuchten. Des Weiteren werden verschieden Gewebe im Lauf des Pflanzenentwicklungszyklus gemessen. Es wurden zum jetzigen Zeitpunkt Proteinexpressionsdaten von fast 16,000 open reading frames (60% des proteinkodierenden Arabidopsis Genoms) gesammelt. Diese werden verwendet um proteomweite, globale und lokale korrelierte Proteinabundanz zu erforschen.

Abb. 3. Breite massenspektrometrische Abdeckung des Arabidopsis thaliana Proteoms.
Abb. 3. Breite massenspektrometrische Abdeckung des Arabidopsis thaliana Proteoms.

Gerichtete Proteomanalytik ist seit langem ein Pendant zu der Discovery-Proteomics in unserer Arbeitsgruppe. Diese wurde vor allem durch akkurate Messung der Peptidfragmentionenmassen mittels des QExactive Plus Massenspektrometers vorangetrieben. Die akkuraten Messungen ermöglichen die Peptididentifizierung und Kartierung der exakten Position posttranslationaler Modifikationen (PTM) mit geringer Fehlerwahrscheinlichkeit. PTM sind für die Proteinfunktion gleichbedeutend wie die Translation der Polypeptidkette selbst. Es werden eine Vielzahl Studien zur quantitativen Messung und Lokalisierung verschiedener PTM durchgeführt, mit einem wachsenden Interesse an Histomodifizerungen und Epigenetik.

Somit erforscht die Arbeitsgruppe die zentralen Fragenstellungen des IPB aus der Perspektive der Veränderungen der zellulären Proteine als Ganzes.

Ausstattung

 

Massenspektrometer

  • Orbitrap Velos Pro (Thermo Scientific)
  • QExactive Plus (Thermo Scientific)

 

HPLC

  • EASY-nLC II (Thermo Scientific)
  • EASY-nLC 1000 (Thermo Scientific)
  • Ultimate 3000 (Thermo Scientific)

 

Software

  • Mascot v.2.5
  • Mascot Distiller
  • SEQUEST
  • Proteome Discoverer v.1.4
  • Progenesis QIP
  • Scaffold 4/ Scaffold PTM 2 Image Quant TL
  • Skyline
  • MaxQuant
  • Perseus
  • MapMan

Das Team

Dr. Wolfgang Hoehenwarter

Mitarbeiter
Abukhalaf, Mohammad Masterstudent
Herr, Tobias Wissenschaftliche Hilfskraft
Proksch, Carsten Technischer Mitarbeiter
Thieme, Domenika Technische Mitarbeiterin

Publikationen nach Tag: Proteomics

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Zeige Ergebnisse 1 bis 10 von 42.

Bücher und Buchkapitel

Mora Huertas, A. C., Schmelzer, C. E. H., Luise, C., Sippl, W., Pietzsch, M., Hoehenwarter, W. & Heinz, A.  Degradation of tropoelastin and skin elastin by neprilysin. Biochimie 146, 73-78, (2018) DOI: 10.1016/j.biochi.2017.11.018.

Neprilysin is also known as skin fibroblast-derived elastase, and its up-regulation during aging is associated with impairments of the elastic fiber network, loss of skin elasticity and wrinkle formation. However, information on its elastase activity is still limited. The aim of this study was to investigate the degradation of fibrillar skin elastin by neprilysin and the influence of the donor's age on the degradation process using mass spectrometry and bioinformatics approaches. The results showed that cleavage by neprilysin is dependent on previous damage of elastin. While neprilysin does not cleave young and intact skin elastin well, it degrades elastin fibers from older donors, which may further promote aging processes. With regards to the cleavage behavior of neprilysin, a strong preference for Gly at P1 was found, while Gly, Ala and Val were well accepted at P1′ upon cleavage of tropoelastin and skin elastin. The results of the study indicate that the progressive release of bioactive elastin peptides by neprilysin upon skin aging may enhance local tissue damage and accelerate extracellular matrix aging processes.

Publikationen in Druck

Al Shweiki, M. H. D. R., Mönchgesang, S., Majovsky, P., Thieme, D., Trutschel, D. & Hoehenwarter, W. Assessment of Label-Free quantification in discovery proteomics and impact of technological factors and natural variability of protein abundance. J Proteome Res. 16 , 1410–1424, (2017) DOI: 10.1021/acs.jproteome.6b00645

We evaluated the state of label-free discovery proteomics focusing especially on technological contributions and contributions of naturally occurring differences in protein abundance to the intersample variability in protein abundance estimates in this highly peptide-centric technology. First, the performance of popular quantitative proteomics software, Proteome Discoverer, Scaffold, MaxQuant, and Progenesis QIP, was benchmarked using their default parameters and some modified settings. Beyond this, the intersample variability in protein abundance estimates was decomposed into variability introduced by the entire technology itself and variable protein amounts inherent to individual plants of the Arabidopsis thaliana Col-0 accession. The technical component was considerably higher than the biological intersample variability, suggesting an effect on the degree and validity of reported biological changes in protein abundance. Surprisingly, the biological variability, protein abundance estimates, and protein fold changes were recorded differently by the software used to quantify the proteins, warranting caution in the comparison of discovery proteomics results. As expected, ∼99% of the proteome was invariant in the isogenic plants in the absence of environmental factors; however, few proteins showed substantial quantitative variability. This naturally occurring variation between individual organisms can have an impact on the causality of reported protein fold changes.

Publikation

Hempel, F., Stenzel, I., Heilmann, M., Krishnamoorthy, P., Menzel, W., Golbik, R., Helm, S., Dobritzsch, D., Baginsky, S., Lee, J., Hoehenwarter, W. & Heilmann, I. MAPKs influence pollen tube growth by controlling the formation of Phosphatidylinositol 4,5-Bisphosphate in an apical plasma membrane domain.  Plant Cell 29, 3030-3050, (2017) DOI: 10.1105/tpc.17.00543

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.
Publikationen in Druck

Kowarschik, K., Hoehenwarter, W., Marillonnet, S. & Trujillo, M.  UbiGate: a synthetic biology toolbox to analyse ubiquitination. New Phytol. (2017) DOI: 10.1111/nph.14900

   Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post-translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process.
    Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning.
    This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression.
    We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time- and cost-effective manner.
Publikation

Huck, N. V., Leissing, F., Majovsky, P., Buntru, M., Aretz, C., Flecken, M., Müller, J. P. J., Vogel, S., Schillberg, S., Hoehenwarter, W., Conrath, U. & Beckers, G. J. M. Combined 15N-labeling and tandemMOAC quantifies phosphorylation of MAP kinase substrates downstream of MKK7 in Arabidopsis. Front Plant Sci 8, 2050, (2017) DOI: 10.3389/fpls.2017.02050

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.
Publikation

Furlan, G., Nakagami, H., Eschen-Lippold, L., Jiang, X., Majovsky, P., Kowarschik, K., Hoehenwarter, W., Lee, J. & Trujillo, M. Changes in PUB22 ubiquitination modes triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 dampen the immune response Plant Cell 29, 726-745, (2017) DOI: 10.1105/tpc.16.00654

Crosstalk between post-translational modifications such as ubiquitination and phosphorylation play key roles in controlling the duration and intensity of signalling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signalling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and thus autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo. 
Publikationen in Druck

Balcke, G., Bennewitz, S., Bergau, N., Athmer, B., Henning, A., Majovsky, P., Jiménez-Gómez, J. M., Hoehenwarter, W. & Tissier, A. F Multi-Omics of tomato glandular trichomes reveals distinct features of central carbon metabolism supporting high productivity of specialized metabolites Plant Cell 29 , 960-983, (2017) DOI: 10.1105/tpc.17.00060

Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics and 13C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO2. Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity. 
Publikation

Strehmel, N., Hoehenwarter, W., Mönchgesang, S., Majovsky, P., Krüger, S., Scheel, D. & Lee, J. Stress-reated mitogen-activated protein kinases stimulate the accumulation of small molecules and proteins in Arabidopsis thaliana root exudates. Front Plant Sci 8 , 1292, (2017) DOI: 10.3389/fpls.2017.01292

A delicate balance in cellular signaling is required for plants to respond to microorganisms or to changes in their environment. Mitogen-activated protein kinase (MAPK) cascades are one of the signaling modules that mediate transduction of extracellular microbial signals into appropriate cellular responses. Here, we employ a transgenic system that simulates activation of two pathogen/stress-responsive MAPKs to study release of metabolites and proteins into root exudates. The premise is based on our previous proteomics study that suggests upregulation of secretory processes in this transgenic system. An advantage of this experimental set-up is the direct focus on MAPK-regulated processes without the confounding complications of other signaling pathways activated by exposure to microbes or microbial molecules. Using non-targeted metabolomics and proteomics studies, we show that MAPK activation can indeed drive the appearance of dipeptides, defense-related metabolites and proteins in root apoplastic fluid. However, the relative levels of other compounds in the exudates were decreased. This points to a bidirectional control of metabolite and protein release into the apoplast. The putative roles for some of the identified apoplastic metabolites and proteins are discussed with respect to possible antimicrobial/defense or allelopathic properties. Overall, our findings demonstrate that sustained activation of MAPKs alters the composition of apoplastic root metabolites and proteins, presumably to influence the plant-microbe interactions in the rhizosphere. The reported metabolomics and proteomics data are available via Metabolights (Identifier: MTBLS441) and ProteomeXchange (Identifier: PXD006328), respectively.
Publikationen in Druck

Frolov, A., Didio, A., Ihling, C., Chantzeva, V., Grishina, T., Hoehenwarter, W., Sinz, A., Smolikova, G., Bilova, T. & Medvedev, S. The effect of simulated microgravity on the Brassica napus seedling proteome. Funct Plant Biol (2017) DOI: 10.1071/FP16378

The magnitude and the direction of the gravitational field represent an important environmental factor affecting plant development. In this context, the absence or frequent alterations of the gravity field (i.e. microgravity conditions) might compromise extraterrestrial agriculture and hence space inhabitation by humans. To overcome the deleterious effects of microgravity, a complete understanding of the underlying changes on the macromolecular level is necessary. However, although microgravity-related changes in gene expression are well characterised on the transcriptome level, proteomic data are limited. Moreover, information about the microgravity-induced changes in the seedling proteome during seed germination and the first steps of seedling development is completely missing. One of the valuable tools to assess gravity-related issues is 3D clinorotation (i.e. rotation in two axes). Therefore, here we address the effects of microgravity, simulated by a two-axial clinostat, on the proteome of 24- and 48-h-old seedlings of oilseed rape (Brassica napus L.). The liquid chromatography-MS-based proteomic analysis and database search revealed 95 up- and 38 downregulated proteins in the tryptic digests obtained from the seedlings subjected to simulated microgravity, with 42 and 52 annotations detected as being unique for 24- and 48-h treatment times, respectively. The polypeptides involved in protein metabolism, transport and signalling were annotated as the functional groups most strongly affected by 3-D clinorotation. 
Publikation

Winkler, M., Niemeyer, M., Hellmuth, A., Janitza, P., Christ, G., Samodelov, S. L., Wilde, V., Majovsky, P., Trujillo, M., Zurbriggen, M. D., Hoehenwarter, W., Quint, M. & Calderón Villalobos, L. I. A. Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction. Nature Commun. 8, 15706, (2017) DOI: 10.1038/ncomms15706

Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.

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