Technologies

Mass Spectrometry

Most of our research is based on high-volume, high-resolution, high-accuracy quantitative bottom-up proteomics data generated by mass spectrometry platform. The MS-Laboratory currently houses five Orbitrap™ mass spectrometers including the latest Orbitrap AstralTM technology, three Orbitrap Eclipse/Lumos Tribrid instruments and one Orbitrap Exploris 480 hybrid mass spectrometer. Each system is coupled on-line to Ultra High Performance Liquid Chromatography systems. In addition to supporting the research projects of the group, our technology is also available as part of scientific collaborations or via the core facility Bavarian Biomolecular Mass Spectrometry Center BayBioMS.

Biochemistry

The questions and challenges posed by the comprehensive analysis of complex proteomes require the development and adaption of various biochemical workflows. Depending on the type of sample and the desired type of analysis we have a large number of standard workflows established in the laboratory, including but not limited to:

• Affinity matrices for subproteome enrichment
• Enrichment strategies for posttranslational modifications
• Thermal proteome profiling
• Different peptide labeling strategies for quantitation and multiplexing
• Fractionation strategies on peptide level

Chemistry and Chemical Proteomics

A major part of our chemical research efforts are directed towards the design and synthesis of novel protein capturing tools. These tools can be peptides or small molecules that we synthesize and immobilize so as to obtain affinity matrices, able to enrich their targets and hence useable for target/partner deconvolution. We are particularly interested in kinases and epigenetic proteins, for which we also dedicate medicinal chemistry projects. Additionally we are developing click and cleave strategies based on triazene linkers to enrich azide-labelled proteins.

The work can be roughly divided in the following area:

• Automated solid-phase peptide synthesis
• Organic synthesis of small molecule protein inhibitors
• Triazene-based cleavable linkers
• Specific and unspecific immobilization on beads

IT and Data Processing

Our IT Infrastructure is tailored to facilitate Mass Spectrometry based proteomics experiments. Raw data collected by individual mass spectrometers are stored on a central file server with larger storage capacity and associated backup systems. Processing along a data pipeline involves e.g. peak processing, protein identification and quantification and is implemented on dedicated hardware including a four-node Mascot cluster and several powerful Windows machines to facilitate data processing with MaxQuant. Computing resources that are not dedicated to such a permanently needed task are made available as virtual machines. Thus, processing power and memory can be balanced and made available where needed. Along the same lines, licensed software is installed on central virtual machines instead of private desktop computers.

Cell biology

The cell biology unit is focused on the preparation of biological samples under defined conditions for proteomic analyses, the development of functional assays to elucidate compound triggered responses in 2- or 3-dimensional in vitro models and the genetic engineering of mammalian cells to precisely monitor the modulation and/or interference of affected signal transduction pathways. For this purpose, we are essentially employing various cell culture systems (e.g., regular stationary or roller culture, large scale culture using a Braun Biostat B fermenter and on-chip culture providing single cell resolution) and monitoring techniques, e.g. manual (IBIDI) or automated live cell imaging (IncuCyte S3). The cell culture facility is complemented by a molecular biology and a microbiology lab. For the analysis of individual cells within a larger population, a Milo™ Single-Cell Western platform is the newest addition to the cell biology facilities.