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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