Proteomics Facility

Proteomics Facility

We provide a complete mass-spectrometry (MS) based bottom-up proteomics infrastructure for the identification and characterization of proteins as well as for the elucidation of their molecular sociology. This includes state-of-the-art nLC-MS/MS experiments employing various data acquisition strategies. We strive to serve all internal investigators by providing both access to proteomics technologies and consultancy advice for running proteomics projects.

Sample inlet of an orbitrap mass spectrometer.

Proteins are a key building block of all living organisms and represent the communication hub in the crosstalk between small molecules (metabolites), extra- and intracellular signals as well as the flow of genetic information (from the genome to RNAs). Hence, together with DNA modifications and non-coding RNA, proteins (and their PTMs) constitute pivotal molecular players in cellular physiology and epigenetic regulation. As a consequence, systematic protein profiling is vital to study cellular homeostasis under regular and disease conditions.

The unit offers a state-of-the-art biomolecular mass spectrometry (MS) analysis service and collaborates with internal research groups in order to develop custom-tailored protein-protein and protein-nucleic acid complex purification and MS analyses strategies to address important biological questions. Our equipment allows us to employ state-of-the-art MS/MS identification and PTM analysis of proteins, enabling the in depth characterization of proteins and protein complexes. Recently, we added targeted MS (PRM: Parallel Reaction Monitoring) as an hypothesis driven proteomics approach offering routine sub-femtomol sensitivity as well as next-generation proteomics via DIA/HRM (Data Independent Acquisition/Hyper Reaction Monitoring) to our portfolio.

Workflow

In the facility, sample processing often starts with tissue/cell lysis, fractionation, digestion of the proteins with site-specific endoproteinases (like trypsin), and peptide cleanup (to remove molecules that interfere with ionization, like detergents, plasticizers, salts). The resulting purified peptide mixture is separated online by C18 reversed-phase chromatography interfaced to a mass spectrometer using nano-UHPLC systems. As the peptides elute from the column, they are ionized by electrospray ionization (ESI) and detected by MS. In the standard data-dependent acquisition (DDA) mode, masses (more precise mass-to-charge values) of intact peptides are detected by MS survey scans, which are followed by peptide fragmentation (MS/MS) to determine the sequence and post-translational modifications of the peptides (in the resulting MS/MS spectral scans).

The unit is very experienced in metabolic labelling of cells (by SILAC), which is the cornerstone for isotope-based quantitative proteomic analyses at the MS1 (survey scan) level. Quantification by isotope-labeled spike-in peptides can also be achieved. TMT labeling as an alternative isotope-based MS/MS quantification workflow, which can be highly multiplexed is currently established. Alternative quantitative approaches are label-free quantification (LFQ) that depend on the sophisticated non-linear alignment of subsequent LC-MS runs.


Selected Publications

1.
Wang Y, Zolotarev N, Yang CY, Rambold A., Mittler G, Grosschedl R (2020)
A Prion-like Domain in Transcription Factor EBF1 Promotes Phase Separation and Enables B Cell Programming of Progenitor Chromatin
Immunity 53(6), 1151-1167.e6
2.
Amol Panhale, Florian M. Richter, Fidel Ramírez, Maria Shvedunova, Thomas Manke, Gerhard Mittler & Asifa Akhtar (2019)
CAPRI enables comparison of evolutionarily conserved RNA interacting regions.
Nature Communications 10, 2682.
3.
Musa YR, Boller S, Puchalska M, Grosschedl R, Mittler G (2018)
Comprehensive Proteomic Investigation of Ebf1 Heterozygosity in Pro-B Lymphocytes Utilizing Data Independent Acquisition
Journal of Proteome Research, 17(1), 76-85.
4.
Kebede AF, Nieborak A, Shahidian LZ, Le Gras S, Richter F, Gómez DA, Baltissen MP, Meszaros G, Magliarelli HF, Taudt A, Margueron R, Colomé-Tatché M, Ricci R, Daujat S, Vermeulen M, Mittler G, Schneider R (2017)
Histone propionylation is a mark of active chromatin
Nature Structural & Molecular Biology 24(12), 1048–1056.
5.
Engelke R, Riede J, Hegermann J, Wuerch A, Eimer S, Dengjel J, Mittler G (2014)
The quantitative nuclear matrix proteome as a biochemical snapshot of nuclear organization
Journal of Proteome Research 3(9), 3940-56.

Find more publications of Gerhard Mittler and his team on PubMed or in the MPG Publication Repository.

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