The Proteomics Core Facility offers a range of techniques for the identification, characterization 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
- Iso-electric focusing of proteins and peptides, using a wide range of pH gradients. Proteins can be separated as a first step in 2D gel electrophoresis.
- High pH (pH 10) reverse phase or SCX chromatography, for offline fraction collection, as a pre-fractionation step in peptide-based proteomics, prior to LC-MS/MS analyses.
- SDS-PAGE gel electrophoresis on gels ranging from 7 to 24 cm. Either in 1D or 2D gel electrophoresis.
- Various gel staining protocols: Coomassie, silver, fluorescent staining by Sypro Ruby or Flamingo, ProQ-Diamond phosphostain.
- Laser fluorescence imaging of fluorescently stained gels.
Protein Digestion and Labeling
- Protein digestion in-gel or in-solution, using a variety of proteases (trypsin, LysC, chymotrypsin, ArgC).
- 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).
- Phosphopeptide enrichment (using TiO2).
- 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).
- Identification of proteins from silver-stained gels (please see here for pre-requisites of sample preparation).
- 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 and HCD.
- 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.
- Protein identification of single proteins by MALDI peptide mass fingerprinting.
- Data analysis for all of the services listed above.
- For protein identification, Mascot is used as the default search engine. Databases can be customized to the user’s needs.
- For protein quantification, we analyse data via MaxQuant or Proteome Discoverer.
- Reports are customised for each user to give the appropriate detail - from a coverage map in Mascot to protein summaries in Excel, to in-depth reporting of quantitative proteome analyses.
Price List (EMBL intranet only).