Background
The new proteomics facility of the MRC Protein Phosphorylation Unit (PPU) is based on the third floor of the Wellcome Trust Building. The facility is shared between Matthias Trost's group, Patrick Pedrioli's group from the Scottish Institute for ceLL Signalling (SCILLS) and the MRC PPU mass spec team which is headed by Dr David Campbell, the facility manager. We share some resources with the proteomics facility of the College of Life Sciences, such as our Mascot server for processing tandem mass spectrometry data.

Contact
For further information please contact Dr David Campbell at (tel. 01382 388347) or Dr Matthias Trost at (tel. 01382 386402).

Technologies
We have the following mass spectrometers:

LTQ Orbitrap classic (Thermo)
Our workhorse. We use it mostly for protein identification and phosphosite analysis by collision induced dissocation (CID) tandem mass spectrometry (MS/MS). It has a Proxeon nano-LC at the front-end.

Orbitrap Velos (Thermo)
Newly installed, this machine will mostly be used for large-scale proteomics and phosphoproteomics analyses. It has a Dionex Ultimate 3000 nano-LC installed.

Orbitrap Velos ETD (Thermo)
Patrick Pedrioli's machine. It has a Proxeon nano-LC at the front-end.

QTrap5500 (ABSciex)
Newly installed, this machine will be used for precursor and neutral loss ion scans to identify phosphopeptides. In addition, we will use it for selected-reaction monitoring (SRM) analyses of phosphopeptides. It has a Dionex Ultimate 3000 nano-LC installed.

amaZon ETD Ion Trap (Bruker)
Newly installed, this machine allows us to do electron-transfer dissociation (ETD) of phosphopeptides on a regular basis. It is also used for protein identification and method development of large-scale analyses. It has a Dionex Ultimate 3000 nano-LC installed.

MALDI TOF-TOF UltrafleXtreme (Bruker)
Newly installed, this machine is used for the identification and characterisation of purified protein and peptide samples as well as for protein assays.

Services

Protein identification

Members of the Unit isolate proteins of interest (usually by SDS-PAGE) and then perform in-gel protease digestions The resultant peptides are analysed by nano LC-MS on the LTQ Orbitrap and the acquired data is processed on an 8 node Mascot server for protein identification.

Protein phosphorylation site identification

Our speciality is the identification of protein phosphorylation sites. We usually identify phosphorylation sites by collision induced dissociation (CID) or electron-transfer dissociation (ETD) in our Orbitrap or amaZon mass spectrometers. However, we also often use the precursor scan on the QTrap to identify the phosphopeptides.

We also perform a number of 32P labelled phosphorylation site analyses. Here, radiolabelled proteins are digested with a protease and the digest separated on an off-line HPLC system which has an on-line radioactivity detector. Peaks are identified by a combination of mass spectrometry and solid phase Edman degradation on an ABI ProCise 494 protein sequencer. This methodology has been used in a large number of publications from the Unit in the past describing novel protein phosphorylation sites and our lab is regarded as one of the world's experts in this technology.

Edman Sequencing

The Edman sequencer is also utilised for N-terminal sequence analyses of proteins after electroblotting onto PVDF membranes. We provide a limited service for researchers outside the MRC PPU who might wish to use this technique

Quantitative proteomics

Members of our facility are also involved in a number of quantitative large-scale proteome and phosphoproteome analyses. We use ProteoProfile (from Pierre Thibault's lab) and Progenesis (Nonlinear Dynamics) for label-free quantitation and MaxQuant (from Matthias Mann’s lab) for SILAC-based experiments.