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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.




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



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



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


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

Publikationen nach Tag: Proteomics

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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.

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. 
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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. 

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.
Publikationen in Druck

Schönberg, A., Rödiger, A., Mehwald, W., Galonska, J., Christ, G., Helm, S., Thieme, D., Majovsky, P., Hoehenwarter, W. & Baginsky, S. Identification of STN7/STN8 kinase targets reveals connections between electron transport, metabolism and gene expression.  Plant J (2017) DOI: 10.1111/tpj.13536

The thylakoid-associated kinases STN7 and STN8 are involved in short- and long-term acclimation of photosynthetic electron transport to changing light conditions. Here we report the identification of STN7/STN8 in vivo targets that connect photosynthetic electron transport with metabolism and gene expression. Comparative phosphoproteomics with the stn7 and stn8 single and double mutants identified two proteases, one RNA-binding protein, a ribosomal protein, the large subunit of Rubisco and a ferredoxin-NADP reductase as targets for the thylakoid-associated kinases. Phosphorylation of three of the above proteins can be partially complemented by STN8 in the stn7 single mutant, albeit at lower efficiency, while phosphorylation of the remaining three proteins strictly depends on STN7. The properties of the STN7-dependent phosphorylation site are similar to those of phosphorylated light-harvesting complex proteins entailing glycine or another small hydrophobic amino acid in the −1 position. Our analysis uncovers the STN7/STN8 kinases as mediators between photosynthetic electron transport, its immediate downstream sinks and long-term adaptation processes affecting metabolite accumulation and gene expression.
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. (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.


Mora Huertas, A. C., Schmelzer, C. E. H., Hoehenwarter, W., Heyroth, F. & Heinz, A. Molecular-level insights into aging processes of skin elastin Biochimie 128-129, 163-173, (2016) DOI: org/10.1016/j.biochi.2016.08.010

Skin aging is characterized by different features including wrinkling, atrophy of the dermis and loss of elasticity associated with damage to the extracellular matrix protein elastin. The aim of this study was to investigate the aging process of skin elastin at the molecular level by evaluating the influence of intrinsic (chronological aging) and extrinsic factors (sun exposure) on the morphology and susceptibility of elastin towards enzymatic degradation. Elastin was isolated from biopsies derived from sun-protected or sun-exposed skin of differently aged individuals. The morphology of the elastin fibers was characterized by scanning electron microscopy. Mass spectrometric analysis and label-free quantification allowed identifying differences in the cleavage patterns of the elastin samples after enzymatic digestion. Principal component analysis and hierarchical cluster analysis were used to visualize differences between the samples and to determine the contribution of extrinsic and intrinsic aging to the proteolytic susceptibility of elastin. Moreover, the release of potentially bioactive peptides was studied. Skin aging is associated with the decomposition of elastin fibers, which is more pronounced in sun-exposed tissue. Marker peptides were identified, which showed an age-related increase or decrease in their abundances and provide insights into the progression of the aging process of elastin fibers. Strong age-related cleavage occurs in hydrophobic tropoelastin domains 18, 20, 24 and 26. Photoaging makes the N-terminal and central parts of the tropoelastin molecules more susceptible towards enzymatic cleavage and, hence, accelerates the age-related degradation of elastin.


Gladilovich, V., Greifenhagen, U., Sukhodolov, N., Selyutin, A., Singer, D., Thieme, D., Majovsky, P., Shirkin, A., Hoehenwarter, W., Bonitenko, E., Podolskaya, E. & Frolov, A. Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics J Chromatogr A 1443, 181–190, (2016) DOI: 10.1016/j.chroma.2016.03.044

Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC–MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC–MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research.


Faden, F., Ramezani, T., Mielke, S., Almudi, I., Nairz, K., Froehlich, M. S., Höckendorff, J., Brandt, W., Hoehenwarter, W., Dohmen, R. J., Schnittger, A. & Dissmeyer, N. Phenotypes on demand via switchable target protein degradation in multicellular organisms Nat Commun. 7, 12202, (2016) DOI: 10.1038/ncomms12202

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.


Sheikh, A. H., Eschen-Lippold, L., Pecher, P., Hoehenwarter, W., Sinha, A. K., Scheel, D. & Lee, J. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana Front. Plant Sci. 7, 61, (2016) DOI: 10.3389/fpls.2016.00061

Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense.

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