Proteomics & Mass Spectrometry

The identification of proteins has been revolutionised by the availability of genome sequences and advances in mass spectrometry. Whilst it is still not possible to catalogue the complete proteome of an organism, the identification of proteins in highly complex mixtures is now tractable. With suitable pre-fractionation of proteins or peptides the in depth analysis of sub proteomes is now a reasonable goal. Furthermore, post-translational modifications, which play a vital role in determining a protein’s function and location, may be determined by mass spectrometry.

We have a hybrid mass spectrometer, the LTQ-Orbitrap from Thermo Fisher Scientific; this was a shared upgrade from the LTQ with the John Innes Centre and the Institute of Food Research.  This machine provides very high mass accuracy typically less than 5 ppm while the fast scan speeds allow great sensitivity. For proteomics we usually use a nano spray system with reverse-phase chromatography.  However, we have a second chromatography system and source which are suitable for higher flow rates. Some metabolite projects are also analysed on the Orbitrap, where the very high mass accuracy can yield a single possibility for chemical composition of small molecules.

We have successfully used several quantitative techniques such asiTRAQ (Applied Biosystems) and metabolic labelling of Arabidopsis cell culture with heavy nitrogen.  We have experience in enriching for phosphopeptides, fractionation of peptides by off-line strong cation exchange chromatograph as well as on-line two phase liquid chromatography separations ( ‘MudPit’). We are currently working to improve the chromatography by using smaller beads (more resolution of HPLC peaks) and smaller columns (lower flow rates mean greater sensitivity).

Services that we provide to Sainsbury Laboratory staff.

  • Advice on design of proteomic experiments
  • Analysis of single proteins or mixtures of proteins by liquid chromatography and mass     spectrometry
  • Interpretation of mass spectrometry data
  • Training in proteomic techniques and analysis
  • Discussion of techniques and ideas for grant and fellowship applications are     encouraged.

    • Links and meetings (internal access only)

    Monthly Protein Forum - All members of SL who work with proteins are encouraged to attend. These meetings are also open to staff and students at JIC and IFR. Please email Alex to get on the email list for dates.

    Wiki pages on mass spectrometry and proteomics

    PhosCalc