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Proteome Analytics

The spatio-temporal remodeling of the proteome, the cellular complement of all proteoforms, is a primary phenotype determinant. As such we are interested in quantifying protein expression dynamics, i.e. the changing abundance, subcellular localization, post translational modification and interaction of proteins in various biological scenarios. It is our goal to gain an understanding of the intricate mechanisms of plant proteome biology.

Fig.1 and 2. Cutting edge mass spectrometry is used to measure peptides and proteins.
Fig.1 and 2. Cutting edge mass spectrometry is used to measure peptides and proteins.

Recently our research group has streamlined and optimized the discovery proteomics approach and adapted it to plants. This technology now allows us to routinely quantify from 6,000 to 9,000 proteins (protein groups) per tissue sample with at least one unique peptide and a peptide and protein FDR threshold of 1%.

A primary research interest of the group is the effects of phytohormones in biotic and abiotic stress adaption. We are applying the deep proteomics strategy in combination with metabolomics and targeted proteomics measurements to shed more light on the interplay of the canonical defense phytohormones salicylic acid, jasmonate and ethylene but also on the role of auxin in the hormone signal signature in reshaping the proteome to resist pathogen attack.

Deep proteomics measurements of various tissues throughout plant development led to date to the accumulation of mass spectrometric evidence of nearly 16,000 protein coding genes which is about 60% of Arabidopsis thaliana open reading frames. This extensive coverage of the Arabidopsis genome is being used to investigate proteome wide correlation of protein abundance in different tissues as well as correlated local protein expression of genes in smaller and larger neighborhoods.

Fig. 3. Deep coverage of the Arabidopsis thaliana proteome
Fig. 3. Deep coverage of the Arabidopsis thaliana proteome

Targeted proteomics approaches are also well established in the group as a complement to discovery proteomics. These were particularly advanced by accurate measurement of fragment ion masses with the QExactive Plus mass spectrometer. This allows interpretation of MS/MS spectra and assignment of PTMs to peptide primary structure with low error probability. Reversible, multi-site PTM has as much an impact on protein function as translation of the nascent polypeptide itself. Numerous directed and undirected proteomics studies that quantify site-specific protein PTM are being performed with a growing interest in histone modification and epigenetics.

Equipment and Instrumentation

 

Mass Spectrometry

  • 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

The Team

Dr. Wolfgang Hoehenwarter

Staff Member
Abukhalaf, Mohammad PhD Student
Herr, Tobias Research Assistant
Proksch, Carsten Technician
Thieme, Domenika Technician

Publications by Tag: Proteomics

Displaying results 1 to 1 of 1.

Printed publications

Teh, O.-K.; Lee, C.-W.; Ditengou, F. A.; Klecker, T.; Furlan, G.; Zietz, M.; Hause, G.; Eschen-Lippold, L.; Hoehenwarter, W.; Lee, J.; Ott, T.; Trujillo, M. Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function BioRxiv (2018) DOI: 10.1101/266171

The exocyst is a conserved hetero-octameric complex mediating early tethering during exocytosis. Its Exo70 subunit plays a critical role as a spatiotemporal regulator by mediating numerous protein and lipid interactions. However, a molecular understanding of the exocyst function remains challenging. We show that Exo70B2 locates to dynamic foci at the plasma membrane and transits through a BFA-sensitive compartment, reflecting its canonical function in secretion. However, treatment with the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We uncovered two ATG8-interacting motifs (AIMs) located in the C-terminal domain (C-domain) of Exo70B2 that mediate its recruitment into the vacuole. Moreover, we also show that Exo70B2 is phosphorylated near the AIMs and mimicking phosphorylation enhanced ATG8 interaction. Finally, Exo70B2 phosphonull lines were hypersensitive to BTH and more resistant to avirulent bacteria which induce SA production. Our results suggests a molecular mechanism in which phosphorylation of Exo70B2 by MPK3 functions in a feed-back system linking cellular signalling to the secretory pathway.

This page was last modified on 14.11.2018.

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