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Diazirine photo-immobilisation for target profiling of natural products and co-factors: pulldown data

ABSTRACT: Finding the targets of natural products is of key importance in both chemical biology and drug discovery, and deconvolution of cofactor interactomes contribute to the functional annotation of the proteome. Identifying the proteins that underlie natural compound activity in phenotypic screens help to validate the respective targets and, potentially, expand the druggable proteome. Here, we present a generally applicable protocol for the photoactivated immobilisation of unmodified and microgram quantities of natural products on diazirine-decorated beads and their use for systematic affinity-based proteome profiling. We show that amongst 31 molecules of very diverse reported activity and biosynthetic origin, 25 could indeed be immobilised. Dose-response competition binding experiments using lysates of human or bacterial cells followed by quantitative mass spectrometry recapitulated <100 µM targets of 9 molecules. Among them, immobilisation of coenzyme A produced a tool to interrogate proteins containing a HotDog domain. Surprisingly, immobilisation of the cofactor flavin adenine dinucleotide (FAD) led to the identification of nanomolar interactions with dozens of RNA-binding proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Cell Suspension Culture, Epithelial Cell, Cell Culture, Escherichia Coli Metabolite

DISEASE(S): Colorectal Adenocarcinoma,Acute Leukemia

SUBMITTER: Polina Prokofeva

LAB HEAD: Prof. Dr. Bernhard Kuster

PROVIDER: PXD033292 | Pride | 2022-10-06

REPOSITORIES: Pride

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	Betulinic_acid_MQ_search.zip	Other
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