Protein interaction networks by proteome peptide scanning.

Area of Science:

• Molecular Biology
• Proteomics
• Biochemistry

Background:

• Protein interactions are crucial for cellular processes, often mediated by small domains binding to short peptides.
• Many low-affinity, transient interactions impact signal transduction but remain unquantified at a proteome-wide scale.

Purpose of the Study:

• To investigate the number of potential interactions per domain and the promiscuity of these interactions across entire proteomes.
• To develop and validate a method for identifying peptide-binding partners for protein domains.

Main Methods:

• Combined phage display and SPOT synthesis to identify and synthesize peptides from the yeast proteome that bind to SH3 domains.
• Probed synthesized peptides with SH3 domains to classify domain specificity and promiscuity.
• Validated identified interactions in vivo using coimmunoprecipitation.

Main Results:

• SH3 domains were grouped into five classes with overlapping specificities but high promiscuity within classes.
• Estimated 6 peptides binding Abp1 SH3 domain (<100 microM) and 50-80 for Yfr024c SH3 domain in yeast.
• Demonstrated the Whole Interactome Scanning Experiment (WISE) strategy is applicable to the human proteome.

Conclusions:

• WISE provides a powerful proteomic tool for rapid, reliable identification of protein interaction partners across diverse proteomes.
• The semiquantitative nature of SPOT synthesis allows for evaluating the physiological relevance of identified interactions.
• This approach facilitates a comprehensive understanding of the cell's interactome by analyzing thousands of interactions simultaneously.