Head of Facility

Dr. Gerhard Mittler
Dr. Gerhard Mittler
Phone:+49 761 5108 -712 / -251

Proteomics Facility

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Service

LC-MS Equipment

The unit is running three hybrid FT-MS instruments that are coupled online, via Proxeon electrospray ionization (ESI) source interfaces to nanobore liquid-chromatography systems (Agilent 1200 nanoLC, Proxeon Easy nLCII, Proxeon nLC1000). One system consists of a Thermofisher LTQ-FT Ultra mass spectrometer that possesses unrivaled mass resolution (greater one million) and mass accuracy (sub-ppm). The second system is a Thermofisher LTQ‑Orbitrap XL+ETD mass spectrometer, which offers excellent resolution (>100,000), mass accuracy (sub-ppm) and high sensitivity, This instrument is equipped with ETD (electron transfer dissociation) and HCD (high collision energy dissociation) capabilities that can give additional structural information useful for de-novo sequencing and post-translational modification (PTM) analysis. The third system is a Thermofisher Q Exactive high-performance benchtop quadrupole Orbitrap mass spectrometer offering very high sensitivity, resolution (140,000), accuracy (sub-ppm) and HCD MS/MS acquisition speed (>10 Hz) making it the instrument of choice for “bottom-up” shotgun proteomics experiments as well as targeted ID and quantification experiments (parallel reaction monitoring).

Prefractionation and sample preparation

For offline protein and peptide chromatography, GE Biosciences SMART and ETTAN-LC microbore HPLC systems are used. Isoelectric focusing (IEF) of peptides is performed on an Agilent 3000 off-gel fractionator. Sample preparation commonly involves in‑gel, in‑solution, FASP or on‑bead trypsin digestion followed by clean‑up using C18 and SAX STAGE tip solid‑phase extraction protocols.

Data Analysis

For peptide and protein identification we use an in-house Mascot database server along with tools from the MSQuant open-source environment. Quantitative proteomics experiments (SILAC and label‑free) are analyzed by MaxQuant and Perseus software (developed in the department of Prof. Matthias Mann at the MPI for Biochemistry).

Core Service

Standard service includes protein identification and peptide mapping (using multiple proteases) from both colloidal coomassie and silver-stained gel bands and low-complexity proteomes (e.g. protein complexes, affinity pull-downs etc.). PTM characterization and quantitative proteomic analysis by either SILAC or label‑free approaches are much more time consuming and are therefore considered as special analysis. Global proteomic projects are evaluated and ranked by an in-house committee whose current members are A. Pichler, R. Grosschedl and G. Mittler.

 
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