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Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+) mobilizing second messenger which triggers Ca(2+) release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca(2+) release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This ‘all-in-one-clickable’ NAADP (AIOC-NAADP) elicited Ca(2+) release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca(2+) release. In mammalian cell homogenates, incubation with low concentrations of [(32)P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [(32)P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [(32)P]5-N(3)-NAADP.