van Rooden_2018_Nat.Protoc_13_752

Reference

Title : Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification - van Rooden_2018_Nat.Protoc_13_752
Author(s) : van Rooden EJ , Florea BI , Deng H , Baggelaar MP , van Esbroeck ACM , Zhou J , Overkleeft HS , van der Stelt M
Ref : Nat Protoc , 13 :752 , 2018
Abstract :

Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. We recently used ABPP to determine the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Whereas our previous protocol described quantification of proteasome activity using stable-isotope labeling, this protocol describes the procedures for identifying the in vivo selectivity profile of covalent inhibitors with label-free quantitative proteomics. The optimization of our protocol for label-free quantification methods results in high proteome coverage and allows the comparison of multiple biological samples. We demonstrate our protocol by assessing the protein interaction landscape of the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The stages of the protocol include tissue lysis, probe incubation, target enrichment, sample preparation, liquid chromatography-mass spectrometry (LC-MS) measurement, data processing and analysis. This approach can be used to study target engagement in a native proteome and to identify potential off targets for the inhibitor under investigation. The entire protocol takes at least 4 d, depending on the number of samples.

PubMedSearch : van Rooden_2018_Nat.Protoc_13_752
PubMedID: 29565900

Related information

Inhibitor DH376    BIA-10-2474    Compound-9368

Citations formats

van Rooden EJ, Florea BI, Deng H, Baggelaar MP, van Esbroeck ACM, Zhou J, Overkleeft HS, van der Stelt M (2018)
Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification
Nat Protoc 13 :752

van Rooden EJ, Florea BI, Deng H, Baggelaar MP, van Esbroeck ACM, Zhou J, Overkleeft HS, van der Stelt M (2018)
Nat Protoc 13 :752