The Proteomics core facility is a high throughput protein analysis laboratory. We focus on protein identification and quantifictaion, structural analysis (post-translational modifications) and protein-protein interactions. We strive to increase the scope, sensitivity and throughput of proteomics technologies and their application to cancer related questions.
The facility helps design 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.
Instrumentation includes three orbitrap mass spectrometers; a QExactive (Thermo), QExactive-HF (Thermo) and Fusion Lumos Orbitrap (Thermo) mass spectrometers all of which are configured with Dionex Ultimate 3000 RSLC nano-HPLC systems. Two off-line separation platforms complement these systems (both Dionex Ultimate 3000 capHPLC systems) for multidimensional chromatography at the protein and peptide level. Data analysis is carried out by core facility staff and/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 focuses and specialises in using stable isotopic strategies (SILAC and TMT) for the relative quantification of protein expression levels and enrichment within protein complexes; currently our multiplexing capacity extends to 11 samples per run. We also develop and run PRM (parallel reaction monitoring assays for targted quantification of panels of proteins.
Specific methods and areas of interest:
Protein profiling of complex biological samples, e.g. tissue, cell extracts
- Protein Profiling by nanoLC/MS
- Multidimensional protein/peptide fractionation by capLC and/or geLC
Targeted protein identification by nanoLC/MS/MS
- Multiple/Parallel reaction monitoring (MRM/PRM analysis)
- Identification of protein-protein interactions from cross-linked immunoprecipitates
- RIME (rapid immunoprecipitation and mass spectrometry of endogenous protein complexes)
- SILAC – stable isotope labelling of amino acids in cell culture
- Isobaric tagging: TMT (tandem mass tags)
- Quantification and sample profiling of modified nucleosides