The Proteomics Core Facility offers a range of techniques for the identification, characterisation and quantification of proteins and proteomes. These include the following techniques and approaches, often used in combination — please enquire for additional applications that may fit your needs.
Protein and Peptide Separation
- High pH (pH 10) reverse phase chromatography, for offline fraction collection, as a pre-fractionation step in peptide-based proteomics, prior to LC-MS/MS analyses
- SDS-PAGE gel electrophoresis
Protein Digestion and Labeling
- Protein digestion in-gel or in-solution, using a variety of proteases (trypsin, LysC, chymotrypsin, ArgC, GluC)
- In-gel acid hydrolysis for determination of N- /C- termini (eg. for samples from limited proteolysis experiments)
- Peptide purification using ZIP tips, Stage tips or OasisHLB μElution plates (Waters)
- Peptide labeling with stable isotopes (TMT, reductive methylation) for relative protein quantification. In addition, we fully support workflows using SILAC labeling
Mass Spectrometry: MS, LC-MS and LC-MS/MS
- Identification of proteins from coomassie stained gels (please see here for MS compatible staining recipe)
- LC-MS (C4 reverse phase UPLC) for protein molecular weight determination under denaturing conditions
- Complex proteome analysis, often combined with additional protein or peptide fractionation techniques (listed above)
- Peptide fragmentation by CID, HCD and ETD
- Relative quantification of proteins and proteomes by stable isotope labeling (SILAC, TMT or dimethyl), using high resolution and high mass accuracy LC-MS and LC-MS/MS
- Identification of phosphorylation and other post-translational modifications on single protein level
- Data analysis for all of the services listed above
- For protein identification, Mascot is used as the default search engine. Databases can be customised to the user’s needs
- For protein quantification, we analyse data via MaxQuant, Proteome Discoverer or IsobarQuant