Proteomics and Mass Spectrometry Team
Proteomics and Mass Spectrometry Team

The proteomics facility of the Unit is based in a purpose built lab on the third floor of the Wellcome Trust Building and is used predominantly by the groups of Matthias Trost, Patrick Pedrioli and Satpal Virdee and by the mass spectrometry service team, headed by 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.

All the research groups within the Unit make significant use of our mass spectrometry service. Mass spectrometry features in over 80 of the MRC-PPU publications over the last five years, demonstrating how critical this technology is to our research. Many of our Units most important discoveries over the years have relied heavily on our mass spectrometry and protein chemistry service. We currently operate 5 mass spectrometers and one protein sequencer that are dedicated to analysing samples generated by researchers in the Unit.

Our mass spectrometry service is overseen by Matthias Trost and day to day operations are directed by David Campbell.

The mass spectrometry service team is made up of
Robert Gourlay (phosphorylation site analysis, especially by using a combination of Mass Spectrometry and Edman sequencing of 32P labelled substrates),
Stella Ritorto ( MALDI-ToF-ToF, MALDI-ISD; offline/online fractionation of complex samples for LC-MS)
Joby Varghese (peptide mass fingerprint analysis by LC-MS and MALDI-ToF-ToF;  phosphorylation site analysis).

These staff not only process the very high volume of samples generated by our Unit, but in addition play a critical role in advising Unit researchers on the best way to generate samples for analysis. They also devote a lot of effort in maintaining all of our mass spectrometry systems in perfect working order and, most importantly, train our Unit staff in how to interpret the mass spectrometry data.

Mass Spectrometry systems

diagram

Thermo Orbitrap Classic / Thermo Proxeon nano-LC
Used for the majority of our protein identifications, by peptide mass fingerprinting; and for our phosphosite analysis, by collision induced dissociation (CID) tandem mass spectrometry.

Thermo Orbitrap Velos  / Thermo Dionex nano-LC
Used mainly for large scale proteomics and phosphoproteomics analysis by those in the Matthias Trost group.

AB Sciex Qtrap 5500  / Thermo Dionex nano-LC 
This is used for primarily for the selected reaction monitoring (SRM) of phosphopeptides

Bruker UltrafleXtreme MALDI TOF/TOF
Used by the service for quality control analysis of expressed proteins, protein assays and analysis of peptides.

Thermo LTQ-Velos-ETD                                 
Used by the service to help pin-point phosphorylation sites and by Daan van Aalten’s group to analyse O-GlcNAc modifications.
           
Additionally, we have a Shimadzu PPSQ33A Protein Sequencer, recently acquired to replace an ABI ProCise, that is used to complement the Mass Spectrometric analysis of 32P labelled peptides as it enables the precise localisation of 32P modified amino acids by use of Edman degradation and N-terminal sequencing.

 

Services

Protein identification
Proteins of interest are isolated by researchers, either by SDS-PAGE and in-gel proteolysis or by in-solution proteolysis of affinity purifications, and  the resultant peptides are analysed by nano LC-MS on the LTQ Orbitrap Classic. We undertake quality control analysis of recombinant proteins generated by our protein production team and for this we use MALDI-ToF-ToF.

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 systems.
We also perform a number of 32P labelled phosphorylation site analyses. Here, radiolabelled proteins are digested with a protease and peptides separated on an off-line HPLC system with an on-line radioactivity detector. Peaks containing 32P are identified by a combination of mass spectrometry and solid phase Edman degradation on a Shimadzu PPSQ33A Protein Sequencer. This methodology has been used in a large number of publications from the Unit describing novel protein phosphorylation sites and our lab is regarded as one of the world's experts in this technology.

Post translational modification identification
This includes analysis for modifications such as Ubiquitylation and occasionally less usual instances such as glycosylation or farnesylation.
All Mass Spec data is processed on an 8 node Mascot server for protein and post translational modification identification, either by Mascot itself or by Proteome Discoverer (Thermo) and its associated phosphoRS software.

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 Unit 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 MaxQuant (from Matthias Mann’s lab) for label-free quantitation and for SILAC-based experiments.

Summary
In general the key questions that our mass spectrometry team analyse concern the precise mapping of phosphorylation and ubiquitylation sites, the protein composition of affinity purifications or immunoprecipitations, quantitative comparisons between samples using SILAC methodology and quality control analysis of recombinant proteins. We are also developing SRM methodology to quantify in vivo phosphorylation sites.

Our work in mass spectrometry is continuing to grow in complexity. We are developing new methods and undertake increasingly intricate mass spectrometry screens to characterise the impact of knock-out/knock-in mutations or drug treatments on global phosphorylation and ubiquitylation. This non-routine analysis is undertaken by dedicated postdocs and PhD students collaborating between the groups of Matthias Trost and Patrick Pedrioli and other Unit PIs.

For all enquires please contact David Campbell (d.g.campbell@dundee.ac.uk) and Matthias Trost (m.trost@dundee.ac.uk ).