When the human genome was sequenced in 2001 about 50 % of genes and corresponding proteins did not have an assigned function. About 20 years later the knowledge has only marginally increased and novel methods to filter the uncharacterized proteome for a streamlined assignment are needed. Given the limited number of drug targets, increasing the scope for drug development especially in the fields of cancer and bacterial infections is an important task. For this purpose, enzymes containing cofactors are prime candidates as they feature conserved chemical moieties and may thus be a convenient starting point for a functional assignment. For example, pyridoxal phosphate dependent enzymes (PLP-DEs) represent a large cofactor class with over 200 reported catalytic activities. The consolidated study of these enzymes bears a unique potential not only to identify novel enzyme members but also select essential proteins as drug targets. This major objectives of our CHEMMINE project is to develop pyridoxal probes which are able to infiltrate bacterial and human cells, get metabolized and finally incorporated in cognate PLP-DEs. These tools are equipped with a bio-orthogonal handle which allows protein enrichment and identification via mass-spectrometry. We seek to utilize this methodology for profiling pathogenic bacteria and cancer cells, report the PLP-ome, select uncharacterized enzymes, unravel their function, monitor the selectivity of pyridoxal-based drugs and design inhibitors against essential proteins. Once successfully established we expand this strategy towards other cofactors and post-translational modifications.