Combining affinity enrichment,cross‐linking with photo amino acids,and mass spectrometry for probing protein kinase D2 interactions

We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by their covalent fixation via UV‐induced activation of incorporated diazirine photoreactive amino acids (photo‐methionine and photo‐leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label‐free LC/MS analysis. Using HeLa cell lysates with photo‐methionine and photo‐leucine‐labeled proteins, we were able to capture and preserve protein interactions that are otherwise elusive in conventional pull‐down experiments. Our approach is exemplified for mapping the protein interaction network of protein kinase D2, but has the potential to be applied to any protein system. Data are available via ProteomeXchange with identifiers PXD005346 (photo amino acid incorporation) and PXD005349 (enrichment experiments).