A whole proteome inventory of background photocrosslinker binding

ABSTRACT: Affinity based protein profiling (AfBPP) is a widely applied methodology for target identification of bioactive molecules. Photocrosslinkers such as benzophenone, diazirine and aryl azide irreversibly link the molecule of interest to its dedicated target protein upon irradiation with UV light. Despite their prevalent application in chemical biology little is known about photolinker-specific off-targets affecting the reliability of results. Here we investigate background protein labelling in intact human cell lines via gel-free quantitative proteomics. Characteristic off-targets were identified for each photo-reactive group and compiled in a comprehensive inventory (photome). In a proof of principle study, H8, an established inhibitor of protein kinase A (PKA), was equipped with a diazirine moiety and revealed, by alignment with the photo-background, unprecedented insight into its in situ proteome targets. Taken together, our findings provide an experimental guideline to surmount photoprobe ambiguity and focus results on biologically relevant binders.

OTHER RELATED OMICS DATASETS IN: MODEL1109130000 MODEL1909260003 MODEL1909260004 MODEL1703310000 MODEL1909260005 MODEL1909260006 MODEL1311110000 MODEL1311110001

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Matthias Stahl  

LAB HEAD: Stephan Axel Sieber

PROVIDER: PXD004009 | Pride | 2016-12-05

REPOSITORIES: Pride