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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)



  • 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

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

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Displaying results 1 to 5 of 5.

Printed publications

Hedtke, T.; Schräder, C. U.; Heinz, A.; Hoehenwarter, W.; Brinckmann, J.; Groth, T.; Schmelzer, C. E. H. A comprehensive map of human elastin cross‐linking during elastogenesis FEBS J (2019) DOI: 10.1111/febs.14929

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross‐linking between hydrophilic lysine‐rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross‐linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post‐translational modifications and cross‐linked peptides by high‐resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross‐link types or remained unmodified. Furthermore, both types of cross‐linking domains, Lys‐Pro and Lys‐Ala domains, participate not only in bifunctional inter‐ but also in intra‐domain cross‐links. We elucidated the sequences of several desmosine‐containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross‐links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys‐Ala domains. In summary, these results provide new and detailed insights into the cross‐linking process, which takes place within and between human tropoelastin units in a stochastic manner.

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.

Schräder, C. U.; Heinz, A.; Majovsky, P.; Karaman Mayack, B.; Brinckmann, J.; Sippl, W.; Schmelzer, C. E. H. Elastin is heterogeneously cross-linked J Biol Chem 293, 15107-15119, (2018) DOI: 10.1074/jbc.RA118.004322

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin’s exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and non-cross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.

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: 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.

Schräder, C. U.; Heinz, A.; Majovsky, P.; Schmelzer, C. E. H. Fingerprinting Desmosine-Containing Elastin Peptides J Am Soc Mass Spectrom 26, 762-773, (2015) DOI: 10.1007/s13361-014-1075-9

Elastin is a vital protein of the extracellular matrix of jawed vertebrates and provides elasticity to numerous tissues. It is secreted in the form of its soluble precursor tropoelastin, which is subsequently cross-linked in the course of the elastic fiber assembly. The process involves the formation of the two tetrafunctional amino acids desmosine (DES) and isodesmosine (IDES), which are unique to elastin. The resulting high degree of cross-linking confers remarkable properties, including mechanical integrity, insolubility, and long-term stability to the protein. These characteristics hinder the structural elucidation of mature elastin. However, MS2 data of linear and cross-linked peptides released by proteolysis can provide indirect insights into the structure of elastin. In this study, we performed energy-resolved collision-induced dissociation experiments of DES, IDES, their derivatives, and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type CxHyN, suggesting that the pyridinium core of DES/IDES remains intact even at relatively high collision energies. The finding of these specific product ions enabled the development of a similarity-based scoring algorithm that was successfully applied on LC-MS/MS data of bovine elastin digests for the identification of DES-/IDES-cross-linked peptides. This approach facilitates the straightforward investigation of native cross-links in elastin.

This page was last modified on 14.11.2018.

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