Updated project metadata. Here we apply functionalized cofactor derivatives to study vitamin B6-dependent enzymes in human cells. We are able to cover for a large fraction of the pyridoxal 5'-phosphate-binding proteome (PLP-ome) and validate our results exemplarily for two PLP-DEs. One of those, named pyridoxal 5'-phosphate binding protein (PLPBP) is characterized further including studies with chemical cross-linking combined with mass spectrometry. We proceed with an in situ PLP-DE target screen applying B6-targeting compounds revealing interesting insights into selectivity. Finally, we broaden our analysis to multiple human cell lines revealing an altered PLP-DE target profile.