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Cancer Research UK Cambridge Institute

 

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Proteomics core facility

The Proteomics core facility focuses on the systematic study of proteins, particularly their structures, interactions and quantification of their expression levels.  The facility is equipped with state-of-the-art instrumentation for CRUK CI researchers requiring access to proteomics technology and expertise.

We provide assitance designing experimental strategies. We also implement and validate previously developed proteomic workflows to profile proteins from diverse biological samples. We also aim to modify or develop entirely new methods and assays when warranted. In addition, we have bioinformatics support for data management and analysis. 

The facility is well equipped with state-of-the-art analytical instrumentation for proteomic studies which includes a range of the Orbitrap mass spectrometers: a LTQ Velos Orbitrap (Thermo), a QExactive and QExactive HF Orbitrap. More recently (Jan 2016) together with the School of Biological Sciences and the Babraham Institute we were successfully applied for a Wellcom Trust Multi-user Equipment grant that funded the purchase of a Fusion Lumos Orbitrap. All platforms are configured to Dionex Ultimate 3000 RSLC nanoHPLC systems. The mass spectrometers are supported by off-line chromatography platforms: two Dionex Ultimate 3000 capHPLC systems for multidimensional chromatography at the protein and peptide level. 1D and 2D gel electrophoresis systems are available from the Research Instrumentation Core facility run by Dr. Jane Gray. Data analysis is supported by an array of bioinformatics and statistical analysis tools.

Specific methods and areas of interest include:

Protein profiling of complex biological samples, e.g. tissue, cell extracts:

  • In-depth proteome analysis using nano LCMSMS Data-dependant acquisition (DDA)
  • Off-line basic pH C18 reversed phase chromatography coupled with nan0LC/MSMS analysis
  • Multi-dimensional GelCMSMS using 1D  or 2D gel formats

Protein:protein interactions

  • Affinity purification of endogenous proteins and their interactiing proteins using Rapid Immunoprecipitation and mass spectrometry of endogenous proteins (RIME);https://www.ncbi.nlm.nih.gov/pubmed/26797456
  • Affinity purification of biotinylated proteins using BioID/APEX2/Avitag technologies.
  • Affinity purification of tagged fusion proteins using standard immunoprecipitation methods.

Targeted protein identification by nanoLC/MS/MS

  • Coomassie and silver stained gel bands of purified proteins
  • In solution digestion of purified proteins
  • Multiple/parallel reaction monitoring (MRM/PRM)of defined peptides

Identification of protein and peptide modifications

  • Phosphoproteomics

Relative quantitation by nanoLC/MS/MS

  • Isobaric Labelling with tandem mass tags (TMT)
  • SILAC - stable isotope labeling of amino acids in cell culture
  • ITRAQ - an isobaric peptide tagging system
  • M/PRM: multiple/parallel reaction monitoring