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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.

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

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			Action	DRS
	20150807_PK_DMSO_H_PK064_L_4.raw	Raw
	20150807_PK_DMSO_H_PK064_L_5.raw	Raw
	20150807_PK_DMSO_H_PK064_L_6.raw	Raw
	20150807_PK_DMSO_H_PK066_L_4.raw	Raw
	20150807_PK_DMSO_H_PK066_L_5.raw	Raw

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