Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification

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.

Development of a Multiplexed Activity-Based Protein Profiling Assay to Evaluate Activity of Endocannabinoid Hydrolase Inhibitors

“… 23 This probe is widely used to study serine hydrolases in complex proteomes. 24 , 25 Although the FP-based probes are known for their broad reactivity, they do not react with all serine hydrolases. 20 Most notably, DAGL-α is among the enzymes which cannot be visualized by FP-based ABPs.…”

Antibiotic production is organized by a division of labour inStreptomyces

“…The dried proteins were dissolved in 0.1% RapiGest SF surfactant (Waters) at 95°C. Protein digestion steps were done according to van Rooden et al 51 . After digestion, trifluoroacetic acid was added for complete degradation and removal of RapiGest SF.…”

“…200 ng of digested peptide was injected and analysed by reverse-phase liquid chromatography on a nanoAcquity UPLC system (Waters) equipped with HSS-T3 C18 1.8 μm, 75 μm X 250 mm column (Water). A gradient from 1% to 40% acetonitrile in 110 min was applied, [Glu 1 ]-fibrinopeptide B was used as lock mass compound and sampled every 30 s. Online MS/MS analysis was done using Synapt G2-Si HDMS mass spectrometer (Waters) with an UDMS E method set up as described 51 .…”

Olaparib‐Based Photoaffinity Probes for PARP‐1 Detection in Living Cells

“…[23] To identify and quantify the bands seen by SDS-PAGE and western blot analysis we used a LC-MS/MS-based label-free quantification (LFQ) method. [16] The results show that PARP-1 is significantly UV-enriched by probe 3, thus indicating that the observed band at 116 kDa can be attributed to PARP-1 ( Figure 3A and B). Competition analysis of significantly UVenriched proteins shows that PARP-1 is outcompeted by olaparib ( Figure 3D); this strongly indicates that the probe binds in the same protein pocket as olaparib.…”

“…[11,12] The ability to monitor ARTDs in vivo is necessary if one wants to study the processes of the drug-target engagement and pharmacokinetics. Small molecule probes that are capable to specifically and covalently bind to their target enzymes in living cells and in tissues have been developed for various classes of enzymes [13][14][15][16][17] and used in successful studies involving in vivo protein detection and quantification. One way to attain such a probe is to convert a known reversible enzyme inhibitor into a covalent probe suitable for affinity-based protein profiling (AfBPP).…”