Sortase A mediated site‐specific immobilization for identification of protein interactions in affinity purification–mass spectrometry experiments

Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein‐protein interactions. Here we make use of the specificity of sortase A transpeptidation reaction to prepare affinity matrices in which a protein bait is covalently linked to the matrix via a short C‐terminal linker region. As a result of this site‐directed immobilization, the bait remains functionally accessible to protein interactions. To apply this approach, we performed SILAC‐based pull‐down experiments and demonstrate the suitability of the approach.