Combining Affinity Enrichment, Cross-Linking with Photo-Amino Acids, and Mass Spectrometry for Probing Protein Kinase D2 Interactions

We present an approach that relies on the affinity enrichment of a target protein from a complex mixture, followed by a UV-induced activation of incorporated diazirine photo-reactive amino acids (photo-methionine and photo-leucine) and a covalent fixation of interaction partners. 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 can be applied to any protein system.