Proteomics core facility
The proteomics core facility provides support with the design, performance and analysis of proteomic studies. In addition we offer specific proteomic methods addressing several areas of research.
We focus on protein identification, structural analysis (post-translational modifications) and protein-protein interactions as well as quantitative assessment of protein expression levels.
Instrumentation includes two orbitrap mass spectrometers, an LTQ Velos Orbitrap (Thermo) and QExactive (Thermo) both of which are configured with Dionex Ultimate 3000 RSLC nano-HPLC systems. Off-line separation platforms are available: two Dionex Ultimate 3000 capHPLC systems for multidimensional chromatography at the protein and peptide level as well as an Akta purifier FPLC. There is capacity for 1D and 2D gel electrophoresis as well as a GE healthcare Typhoon Trio+ imager available in the Research Instrumentation core facility. Data analysis is carried out by core facility staff or using statistical software packages supported by the Bioinformatics core facility. In addition to proteomic applications we exploit the high resolution and mass accuracy of both Orbitraps to quantify known and discover novel modified nucleosides from genomic DNA.
The core aids researchers designing experimental strategies. We implement and validate previously developed proteomic workflows to profile proteins from diverse biological samples as well as developing entirely new, bespoke methods and assays when required.
We perform full proteome and targeted analyses. The core focuses and specialises in using stable isotopic strategies (SILAC and TMT) for the relative quantification of protein expression levels. Currently our multiplexing capacity extends to 10 samples per run.
Specific methods and areas of interest:
Protein profiling of complex biological samples, e.g. tissue, cell extracts
- Profiling by nanoLC/MS
- Multidimensional protein/peptide fractionation by capLC and/or geLC
Targeted protein identification by nanoLC/MS/MS
- Multiple reaction monitoring (MRM analysis)
- Coomassie and silver stained gel bands of purified proteins
- In solution digestion of purified proteins
- Identification of protein-protein interactions from cross-linked immunoprecipitates
- RIME (rapid immunoprecipitation and mass spectrometry of endogenous protein complexes)
Relative quantitation by nano LC/MS/MS
- SILAC – stable isotope labelling of amino acids in cell culture
- Isobaric tagging: TMT (tandem mass tags)
- Quantification and sample profiling of modified nucleosides