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Photoaffinity Labeling in Drug Discovery Research

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Photoaffinity Labeling for Structural Probing Within Protein

Abstract

Photoaffinity labeling (PAL) is a method that forms an irreversible covalent bond between photoreactive ligands and neighboring amino acids under the irradiation of light. PAL is utilized in the pharmacological and biochemical identification of ligand target molecules and ligand binding sites. Recent technological advances in mass spectrometry have enabled measurement of the mass of intact proteins and peptides with extremely high accuracy. Mass spectrometry has also been adopted in PAL to analyze labeled proteins and identify crosslink amino acid, although this application has been mostly for soluble proteins and reports on the successful identification of crosslink amino acids in GPCRs by mass spectrometry are scarce. In this chapter, we describe in detail our PAL technique that determines crosslink amino acid using the human adenosine A2A receptor as a representative class A GPCR.

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Acknowledgments

The authors are grateful to Ichio Shimada (Tokyo University, Japan) for providing the cDNA of the hA2AAR and his technical advice on protein production. We thank Yasumaru Hatanaka (Toyama University, Japan) for his advice on PAL experiments. We warmly thank Toshiki Honma for FP data analysis, Shigeru Yonekubo for HPLC analyses, and Yoshinori Nonaka for NMR measurements. We also thank Kosuke Okazaki (Japan Agency for Medical Research and Development, AMED, Japan) for his insights.

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Authors and Affiliations

  1. Central Research Laboratories, Kissei Pharmaceutical Co., Ltd., 4365-1 Kashiwabara, Hotaka, Azumino, Nagano, 399-8304, Japan

    Hideyuki Muranaka, Takaki Momose, Chiaki Handa & Tomonaga Ozawa

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  1. Hideyuki Muranaka
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  2. Takaki Momose
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  3. Chiaki Handa
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  4. Tomonaga Ozawa
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Correspondence to Hideyuki Muranaka .

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Editors and Affiliations

  1. University of Toyama, Toyama, Japan

    Yasumaru Hatanaka

  2. Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan

    Makoto Hashimoto

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Muranaka, H., Momose, T., Handa, C., Ozawa, T. (2017). Photoaffinity Labeling in Drug Discovery Research. In: Hatanaka, Y., Hashimoto, M. (eds) Photoaffinity Labeling for Structural Probing Within Protein. Springer, Tokyo. https://doi.org/10.1007/978-4-431-56569-7_12

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