Prof. M. Krüger/Dr. C. Frese: Quantitative Proteomics (CMMC/CECAD)

MS-based proteomics is routinely used to investigate the composition and dynamics of cellular
organelles, protein complexes, and signaling pathways. Contemporary shotgun proteomics
utilizes site-specific proteases to perform protein digestion and the resulting peptide mixture is
then subjected to mass spectrometery. Modern high resolution mass spectrometers, such as
quadrupole Orbitraps, consist of a selection quadrupole, a high-efficiency C-trap and higherenergy
collisional dissociation (HCD) octopole collision cell that allows for a rapid and sensitive
fragmentation of peptides.
Another challenge for proteomics is the identification of peptides with post-translational
modifications (PTMs) from cells and tissues since biological relevant modifications are often of
low abundance. To identify those PTMs by MS, several modification enrichment strategies were
developed.
1) Quantitative proteomics (using LFQ, SILAC, and ITRAQ): The proteomics facility at the
CECAD uses metabolic and chemical labeling techniques for protein quantification in cell
culture and living animals. Protocols and advice for metabolic and chemical labeling are
available and the technical assistant of the CF will support members of the research training
group with SILAC labeling, protein extraction, and protein/peptide separation.
2) Protein-protein interaction studies: Protein-protein interactions are fundamental to the
understanding of biological processes. This method is based on the enrichment of bait
proteins using a specific antibody (endogenous level) or in the case of a tagged protein
(FLAG, HA, or GFP), a tag-specific antibody that is immobilized on a surface (e.g. beads,
well plates). Bait proteins and their interactors (preys) become enriched after applying
several mild washing steps. The protein mixture is then analyzed by liquid chromatography
tandem mass spectrometry (LC-MS/MS) and quantified by an intensity-based label-free
quantification.
3) PTM mapping - Enrichment of post-translational modifications: The major challenge for
the detection of PTMs is the low stoichiometry for most of the modified peptides in complex
biological samples. The enrichment of phosphopeptides will be performed by high-pH
reversed-phase chromatography and phosphopeptide extraction with titanium dioxide beads.
For the enrichment of ubiquitination sites (ubiquitin remnants), tryptic peptides from
cells/tissue will be immunoprecipitated with an ubiquitin remnant motif antibody and then
analyzed by mass spectrometry.
Internal resources: The CF consists of one LTQ Orbitrap discovery and three high resolution
hybrid quadrupole Orbitrap mass spectrometers (2xExactive Plus, QExactive Plus HF-X). Each
instrument is connected to a nano-HPLC system and uses the electro-spray ionization technique
(ESI). Gel- and liquid-based separations on the protein and peptide levels are performed using
1D-SDS PAGE, isoelectric fractionation, as well as anion exchange and reversed phase
chromatography (UHPLC).