High throughput analysis of protein interactions and modifications using peptide arrays

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Signal transduction in metazoans is effected via the dynamic organization of protein interaction networks. These interactions are determined by the affinity and the specificity of the protein partners in a given cell. Post-translational modifications (PTMs) may create or preclude protein-protein interactions. In particular, phosphorylation and acetylation are two of the most frequent PTMs in eukaryotes. These modifications are of particular importance in cell signalling, as they represent stable alterations of protein structure and function, yet ones that may be rapidly controlled through the opposing action of enzymes to regulate the formation of new signalling complexes, or induce changes in existing complexes, via the recruitment and/or activation of a collection of downstream signalling molecules.******The applicants Bisson, Elowe and Lambert are early- and mid-career investigators who have a strong expertise in cell signalling, in particular with protein-protein interactions and PTMs. All three have developed NSERC-funded research programs that rely heavily on proteomics, protein biochemistry and cell biology. These research programs would tremendously leap forward with the addition of high-throughput peptide screening in this toolkit. The requested equipment is an Intavis ResPep SLi peptide synthesizer, combined with an automated SPOT synthesis module, to allow custom peptide array synthesis. ******The ResPep SLi is a fully automated benchtop instrument dedicated to solid-phase peptide synthesis. In the requested configuration, it allows for the parallel synthesis of up to 96 peptides of 10-30 residues in a 1-10 µmol scale. We also request an automated SPOT synthesis module, which may generate custom peptide arrays via repeated deposition of activated amino acids as spots on a cellulose membrane. Two membranes each containing up to 600 peptide spots may be synthesized in parallel on freely defined grids, in a single day. These peptide arrays allow for the rapid screening of hundreds of peptides in a single experiment, in order to perform epitope mapping, analysis of kinase substrates, receptor binding studies and peptide-nucleic acid arrays. The lack of an accessible peptide array synthesizer is a major bottleneck that delays the progress of the applicant's research programs, as these high-throughput approaches are essential for growth of the Bisson, Elowe and Lambert research programs.******The cutting-edge peptide synthesizer and SPOT module, combined with infrastructure available within our Institute (e.g. mass spectrometry platforms), will expose HQP to a plethora of techniques that will allow them to develop a highly sought technical expertise in areas that are in dire need of qualified personnel such as high-throughput screening and proteomics. The proposed instrument will accelerate research training and will provide trainees with a solid base to perform innovative research.