Updated project metadata. We have devised a simple and robust LC-MS/MS compatible protocol for subcellular protein localization studies. Here, we combine it with in depth proteome profiling and utilize the resulting dataset to predict the subcellular localization of more than 12000 proteins in 5 human cancer cell lines. The data allows for exploration of both cell line specific and perturbation related protein localization changes as well as domain and splice variant effects and protein complex formation. The data is made accessible through the online resource www.subcellbarcode.org.