Edit

Proteomics Core Facility

This facility provides a full proteomics infrastructure for the identification and characterisation of proteins.

Infrastructure in this facility is centered around state-of-the-art mass spectrometry for MS and LC-MSMS experiments. This is complemented by chromatographic and electrophoretic systems for protein and peptide separation.
Edit

Mikhail Savitski

Team Leader and Head of Proteomics Core Facility

mikhail.savitski@embl.de

ORCID: 0000-0003-2011-9247

Edit

The Proteomics Core Facility provides a full proteomic infrastructure for the identification and characterization of proteins. This includes various platforms for protein and peptide separation, and state-of-the-art mass spectrometry for MS and LC-MS/MS experiments.

This website intends to give guidelines for sample submission to the PCF, and to provide practical information on sample preparation. In addition, background information on several aspects of mass spectrometry and proteomics is available in a collection of websites and review articles. Should this not give you the information you are looking for, staff in the PCF are always available to answer your questions and advise on sample preparation.

Services

Proteomics:

Protein identification from gel or in-solution
High resolution and high mass-accuracy MS, MS/MS, and LC-MS/MS (Thermo Orbitrap Velos Pro, Q-Exactive plus, Fusion Lumos) for identification and quantification of proteins in complex mixtures
Protein quantification by stable-isotope labeling (SILAC, TMT and dimethyl labelling)
Identification of post-translational modifications on purified proteins
Multi-dimensional peptide separation (liquid chromatography)

Analysis of intact proteins:

Molecular weight determination of intact proteins by ESI mass spectrometry
Determination of N- and C-termini of proteins and products of limited proteolysis
Verification of incorporation of non-natural amino acids

Techniques and approaches

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:

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, AspN).
In-gel acid hydrolysis for determination of N- /C- termini (eg. for samples from limited proteolysis experiments).
Peptide purification using OasisHLB μElution plates (Waters), Sep-Pak C18 cartridges (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 customized to the user’s needs.
For protein quantification, we analyse data via MaxQuant, Proteome Discoverer or IsobarQuant.

Please enquire for additional applications that may fit your needs.

Links and tutorials

Here you can find background information on the principles of mass spectrometry and proteomics.

This includes some instructive websites, as well as a collection of review articles that we have put on our bookshelf.

Services fees

Consumables	EURO €
Desalting (per sample)	20
TMT6/11-plex labelling reagent	140
TMT16-plex labelling reagent	280
High pH fractionation (per sample)	20
Peptide-SEC (per sample)	20

Measurement time (per hour)	EURO €
Academia	90
Industry	110

In order to estimate the total price for a specific type of a proteomics experiment such as, for instance, a full proteome analysis, please contact the PCF (pcf@embl.de) to discuss.