DKTK Core Facilities: Proteomics

Proteomics Facility München

The Core Facility Proteomics at the DKTK Partner site comprises five high-end mass spectrometers, several liquid chromatography systems and terabyte scale computing infrastructure.

The range of supported applications span quantitative protein expression profiling of human and animal tissues, tumors, cell lines and body fluids, dynamic analysis of post-translational modifications, protein-protein interactions, drug target identification, drug mechanism of action analysis and many more. We can offer discovery type proteomic experiments using multidimensional chromatography coupled to the latest Orbitrap technology as well as targeted assays using parallel reaction monitoring. All major quantification workflows are established in the laboratory including SILAC, TMT and a range of label-free analysis. We typically work on the basis of scientific collaboration in which we engage our expertise along the entire path from experiment design through to data acquisition and bioinformatic analysis.

Proteomics Core Facility Frankfurt

The Proteomics Core Facility at the Frankfurt location is an initiative of the DKTK and is equipped with three Q Exactive mass spectrometers from Thermo Fisher (one Q Exactive, one Q Exactive Plus and one Q Exactive HF instrument). The available technology can be used to e.g. identify and quantify protein expression patterns, post-translational protein modifications such as phosphorylation, acetylation and ubiquitination, and protein complexes.

This makes it possible to conduct a comprehensive characterisation of oncogenic mechanisms at the protein level, which includes mapping of oncogenic signal transduction processes. The technical advances made in recent years mean that it is now possible to identify and quantify in relative terms several thousand proteins from just a few micrograms of protein. The main activities of the Proteomics Core Facility relate to translational research topics in the field of acute leukaemia, lymphoma, lung cancer, rectal cancer and brain tumours. The research uses cell culture models and freshly isolated tumour cells, as well as fresh frozen and formalin-fixed tissue samples. All the procedures necessary for sample preparation are established at the Proteomics Core Facility and are available to users. For the relative quantification of protein samples, the methods available include stable isotope labelling (SILAC), iTRAQ and label-free methods. Absolute protein quantification is also possible. Besides biochemical analysis, the Proteomics Core Facility is also used to conduct bioinformatic analyses of mass spectrometry data, making it possible to provide DKTK scientists with complex datasets once the data has been suitably processed.

Cancer Metabolomics Platform Berlin

The metabolome reflects the current functional status of the cell. Metabolomic changes play a central role in the pathogenesis and therapy of malignant diseases. The at the DKTK partner site Berlin established gas chromatography, coupled with high resolution mass spectrometry (GC-MS), is a sensitive and robust platform for the analysis of metabolites of the primary metabolism (amino acids, sugars, nucleic acids) (Fig. 2). This technique offers a broad applicability in tumor biology and interdisciplinary problems. The MS technology (APCI Q-TOF) allows quantitative statements and is particularly suitable for the elucidation of the structure of unknown analytes and for the analysis of very small samples volumes. Metabolic flux analysis is currently under establishment. As increasingly important extension of other, in Berlin reproached omics platforms, the cancer metabolomics platform enables functional, systems biology insights into the tumor metabolism and contributes to the identification of novel therapeutic principles.

Genomics and Proteomics Heidelberg

The DKFZ Genomics and Proteomics Core Facility (GPCF) is a central research infrastructure providing DKTK members access to sophisticated key technologies that serve the need of biomedical science and offering high quality solutions at reasonable costs. Trained and dedicated personnel ensure highest quality and reproducibility of our services. Services are processed according to the "first come - first served" principle.

The GPCF technology portfolio mostly covers the following areas:

• The genomics part of the facility operates one of Europe’s largest next-generation sequencing units offering Illumina HiSeq and MiSeq technologies, the latest Illumina Hi-Seq X-Ten platform and the NovaSeq und NextSeq technologies. GPCF performs WGS, WGBS, WES, ChIP-seq, mRNA-seq, smallRNA-seq etc. within several national and international cancer genome projects (e.g., INFORM, DKTK), in addition to a variety of individual in-house projects. The unique Illumina HiSeq X-Ten platform for whole genome sequencing, which was funded by DKTK and established in the years 2014-2015 allows whole genome sequencing of more than 8,000 patients per year.

  Gene and miRNA expression profiling (Illumina, Agilent, Affymetrix), genotyping (Illumina, Affymetrix) and methylation (Illumina MethylationEPIC BeadChip) analysis is carried out using array-based technologies. Moreover, an array-based kinase activity profiling service is established. At the beginning of January 2019, the High Throughput Sequencing-Unit and the Microarray applications for clinical analysis were accredited according to the international standard DIN EN ISO/IEC 17025.

• The proteomics section provides technologies for analyzing proteins, protein modifications and small molecules by state-of-the-art mass spectrometry and NMR in a qualitative and quantitative manner.
• In the field of functional genomics the GPCF generates monoclonal antibodies on demand, some of which are by now used in diagnostics. The service covers the production of monoclonal and polyclonal antibodies, as well as the non-GMP production and purification of antibodies on a larger scale for preclinical studies.
  Isogenic cell lines stably expressing recombinant proteins or shRNAs from a particular genomic locus are generated e.g., to assess phenotypes of proteins and mutants in in vitro as well as in vivo systems, e.g., in xenograft mouse models. Furthermore, a lenti/retrovirus transduction service, which includes filing the application, risk assessment, cell line transduction and the complete safety documentation, is established.
• The central Infrastructure of the GPCF offers assisted access to several costly instruments (e.g., qPCR- and ddPCR-system) being utilized by a number of scientific groups with help of an online booking system, and it further distributes the human ORF clone resource of the international ORFeome Collaboration (>17,000 human genes) for recombinant expression of encoded proteins, also in isogenic cell lines.

Our mission is to always enable application of state-of-the-art technologies and to offer high quality solutions. We carry out joint projects with scientific groups within DKFZ to implement the latest technologies that serve the needs of science. In addition to providing full or assisted access to services, we are involved in the DKFZ teaching program, and organize tech-talks, seminars and practical courses where we inform interested scientists, predocs and technicians on the technological background of applications.

GPCF Homepage: www.dkfz.de/gpcf