Proteomics

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Olaparib based photo-affinity probes for PARP-1 detection in living cells


ABSTRACT: The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes poly adenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to DNA damage sites. Inhibition of PARP activity by olaparib can cause cell death which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work we present the design, synthesis and biological evaluation of photo-activatable affinity probes inspired by the olaparib molecule which are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS-PAGE, western blotting and label-free LC-MS/MS quantification analysis show that the probes target the PARP-1 protein and are selectively outcompeted by olaparib suggesting binding in the same enzymatic pocket.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Bogdan Florea  

LAB HEAD: Bogdan Iulius Florea

PROVIDER: PXD018661 | Pride | 2020-04-29

REPOSITORIES: pride

Dataset's files

Source:
Action DRS
20191125_JV_DMSO_01.zip.raw Raw
20191125_JV_DMSO_02.zip.raw Raw
20191125_JV_DMSO_03.zip.raw Raw
20191125_JV_DMSO_04.zip.raw Raw
20191125_JV_OLPRB-003_01.zip.raw Raw
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Publications

Olaparib-Based Photoaffinity Probes for PARP-1 Detection in Living Cells.

Voorneveld Jim J   Florea Bogdan I BI   Bakkum Thomas T   Mendowicz Rafal J RJ   van der Veer Miriam S MS   Gagestein Berend B   van Kasteren Sander I SI   van der Stelt Mario M   Overkleeft Herman S HS   Filippov Dmitri V DV  

Chembiochem : a European journal of chemical biology 20200513 17


The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes polyadenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to sites of DNA damage. The inhibition of PARP activity by olaparib can cause cell death, which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work, we present the design, synthes  ...[more]

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