Probing the recognition of post-translational modifications by combining sortase-mediated ligation and phage-assisted...

Reversible post-translational modifications (PTMs) are key regulators of protein function and modulate a multitude of protein-protein interactions in signal transduction networks. Here, we describe a strategy for determining the modification preferences of PTM-binding proteins with only minimal protein amounts that can be obtained by immunoprecipitation from mammalian cell lysates. This method bases on the combination of sortase-mediated ligation and phage-assisted selection strategies. This method can be used to analyze the type of modification that mediates the interaction as well as the influence of the amino acids flanking the modification sites. We have demonstrated the applicability of this method by probing the interaction of phosphorylated tyrosine and serine residues with their respective binding domains.