Nat. Methods http://dx.doi.org/10.1038/nmeth.3998 (2016)

The addition of nitric oxide promotes the release of intracellular zinc, which can promote toxicity if unmanaged. Excess zinc is thought to be sequestered in cytoplasmic vesicles; however, a detailed characterization of these vesicles has remained elusive. Miki et al. developed a chemical proteomic platform to identify proteins present under high zinc levels in cells. For this, they used the protein labeling reagent AIZin, which contains a zinc-binding site (dipicolylamine), an acyl imidazole electrophilic reactive group and a fluorophore. AIZin is activated only in the presence of zinc, which allows zinc-responsive protein labeling. The addition of AIZin to glioma cells in the presence of the NO donor resulted in the detection of fluorescent bands that were depleted in the presence of a Zn chelator. Subsequent LC-MS/MS analysis revealed an enrichment of endoplasmic reticulum (ER) and vesicle proteins three hours after NO treatment, suggesting the presence of zinc vesicles. They confirmed that seven candidate proteins including calreticulin and Rab1A exhibited overlapping expression with zinc vesicles in cells. Given the known localization of the identified proteins, the authors propose that the zinc-rich vesicles may be in transit between the ER and Golgi. Although it remains to be determined whether these proteins are involved in the formation of the zinc vesicles, this approach provides a facile tool for the identification of proteins involved in zinc homeostasis on a proteome-wide scale.