Johann Holzmann, Postdoc

Proteins aren’t lonely players; rather they form a highly dynamic network of labile and/ or stable interactions. One major goal in the characterisation of a protein complex is the knowledge of its abundance and stoichiometry. We developed a novel method (termed Equimolarity through Equalizer Peptide or EtEP) that permits accurate and cost-efficient absolute quantification and subsequent stoichiometry determination of protein complexes according to the isotope dilution mass spectrometry principle. Using our EtEP strategy we are determining the absolute concentration and stoichiometries of protein complexes that play crucial roles in cell cycle regulation.

Peter Pichler and Thomas Köcher, Postdocs

Proteins regulated in disease may provide insight into the molecular mechanisms of disease processes and might eventually serve as candidates for clinically useful biomarkers. During the last decade there has been a growing interest in the description of biological systems in a quantitative and systematic manner. In parallel, methods have been developed for quantifying and identifying proteins in an unbiased way. One of these methods, termed isobaric tags for relative and absolute quantitation (iTRAQ), has become increasingly popular as the approach is applicable not only to cell culture but also to animal or human tissue. The technique can be used in conjunction with different mass spectrometers and fragmentation techniques. We have contributed to the further improvement of iTRAQ methodology, and we have developed a broadly applicable and precise quantitative analytical strategy based on iTRAQ which was successfully applied to several projects: A mouse liver hepatitis model utilizing c-jun knock-out mice, models of heart failure and brain disease in mice, and a brain tumor model in flies. We have also applied iTRAQ to the analysis of protein complexes under different conditions to elucidate their dynamic behavior.

Karin Großteßner-Hain, Postdoc

The protein kinase Plk1 is a key regulator of mitotic progression and cell division in eukaryotes. Normal cell division is therefore extremely sensitive to Plk1 levels and activity. Because proliferating cells such as tumor cells are dependent on cell division very much as well and Plk1 inhibition stops the cells from dividing, the pharmaceutical industry is pursuing the generation of Plk1 inhibitors as anti-cancer drugs. Until now, only a few target sites for Plk1 are known, and assays to identify Plk1 substrates have largely been carried out in vitro, without the context of cellular regulatory systems. In this project we describe an unbiased proteome-wide approach for the discovery of Plk1-regulated phospho-sites in human cells, based on the use of a specific Plk1 inhibitor and quantitative mass spectrometry. The experimental set up includes the labeling which was introduced by esterification of the peptides from the two conditions (+/- Plk1 inhibitor) with isotopically-distinct forms of methanol. Afterwards the sample was fractionated via strong cation exchange chromatography (SCX) employing a salt/pH gradient. The final step was the phosphopeptide enrichment with immobilized metal affinity chromatography (IMAC).The results presented in this project could provide the basis for many future biological and biochemical experiments to understand how Plk1 functions in the cell, as well as providing potential biomarkers for clinical and diagnostic purposes. Further we can use the potential Plk1 substrates for the refinement of the reported Plk1 consensus motif.

Thomas Taus, Diploma Student

In order to identify the underlying biological pathways, we optimized strategies to identify numerous protein phosphorylations within complex samples. Nevertheless, some uncertainty in the exact localization of the phospho-moiety exists for a significant number of phospho-peptides which is due to the presence of multiple putatively phosphorylated amino acids, such as serine, threonine, or tyrosine. Simple database search, as often used for peptide identification, is therefore not enough for phospho-site validation. To overcome laborious manual inspection of the spectra, we developed a software tool which ranks all putative phospho-peptide sequences according to their overlap with MS/MS data.

Debora Broch-Trentini, PhD student; Andreas Schmidt, PostDoc

Decades of research have established protein phosphorylation as a universal mechanism for cellular control and adaptation. It is difficult to come across a signaling pathway that is not affected at some level by this type of modification. Accordingly, protein phosphorylation analysis has become one of the major focuses in the mass spectrometry field. Today, there is a wealth in information concerning phosphorylation of serine, threonine and tyrosine residues. Much less explored is this modification for histidine, arginine and lysine, in which the phosphate group is attached to a side chain nitrogen atom, forming a phosphoramidate bond that is unstable at acid pH – a condition commonly employed in standard proteomic protocols. The first reports of the so called N-phosphorylation date back to the 60 and 70 decades, and since then this theme has been scarcely addressed. In cooperation with the group of Tim Clausen (IMP), we recently described arginine-phosphorylation as post-translational modification of the bacterial transcription factor CtsR which impaired its DNA binding abilities. This might be an example for the importance of N-phosphorylation for transcriptional regulation and other processes that involve protein-DNA binding. We are interested in taking advantage of recent advances in mass spectrometry to develop and employ tools for the systematic investigation of the occurrence of N-phosphorylations in living cells, with special attention to arginine-phosphorylation in bacteria and eukaryotes.