Work Package 7: Mapping Protease Cleavage Specificities – University of Copenhagen

HIPAD > The Project > Work Package 7

Work Package 7:

  • Provide analytes of interest (primarily of immune origin) and demonstrate the power of using an integrated peptide microarray, label-free detection and bioinformatics approach to analyze their specificities.
  • Provide target proteins for the development of peptide microarray-based approach to the identification of peptide probes

Recombinant human MHC class I and II molecules, will be generated and offered to peptide microarrays where binding kinetics will be examined (e.g. using SPRi). For MHC class I molecules that prefer peptides with free amino and carboxy terminals, the peptides will be tethered in situ to the peptide support using an internal amino acid leaving the desired free terminals.

A vey ambitious (high-risk) goal will be to get a T cell receptor to bind to peptide microarray-MHC complexes. T cell receptors from a single clone will be oligomerised and offered to peptide microarrays that have been pre-coated with the relevant MHC restrictions element.

Various antibody preparations (monoclonal, polyclonal) directed against linear or discontinuous protein
epitopes will be prepared and offered to peptide microarrays. The peptide microarrays will express various representations of target antigens (from exhaustive truncation analysis of known epitopes over amino acids scanning to ab initio approaches using large random libraries and systematic analysis of leads here from).

We have access to a large repository ( of purified recombinant proteins, which we will use to test the hypothesis that peptides can be used as probes. High-density highly diverse peptide microarrays will be used to search for leads, which will be expanded to identify specific high-affinity peptide probes. 
In all cases, bioinformatics will be used to analyze the data and design new peptide microarrays aimed at expanding the fine specificity mapping. In an attempt to detect cross-reacting self-epitopes for antibodies (as well as T cell receptors) we will use bioinformatics predictors to predict potential auto-epitopes from the entire proteome.

Partner responsibilities:

  • P1 contributes with reagents (i.e. MHC), designs and analysis of peptide-analytes
  • P2 generates peptide microarrays
  • P3 performs MALDI IMS analysis
  • P4 is responsible for performing SPRi analysis
  • P10 conducts the for bioinformatics analysis