Tag-creation approaches for highly efficient profiling of interacting proteins and domains

Diazirine-based photoaffinity labeling is recognized as one of the most reliable methods for identification of biomolecular interactions because of its excellent chemical and physical properties. To avoid time-consuming steps in the analysis of a tiny amount of labeled product, functionalization of photoprobe should be an essential subject in this method. However, addition of functions often affects affinity of bioactive molecule. In this chapter, multifunctional diazirine-based photocross-linkers and their strategies for rapid target protein profiling of bioactive molecules are described, especially tagging methods after cross-linking including post-labeling using cleavable function, tandem labeling using clickable function, and fluorogenic labeling. Further, a unique target-visualization strategy is presented for facile identification of labeled site within protein using isotope-coded fluorescent tag, which can easily distinguish the target from the enormous range of biomolecules in analytical process using LC-MS/MS. Without any chemical treatments, a coumarin tag is photochemically generated on ligand-interacting surface of protein through structural change from nonfluorescent photocross-linker unit with accompanying cleavage of ligand molecule.