Dataset Information

SITE-SPECIFIC ACTIVITY-BASED PROTEIN PROFILING USING PHOSPHONATE HANDLES

ABSTRACT: Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses IMAC-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe (ABP), enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID–ABPP (Activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated Afatinib derivative (PF-06672131). As PhosID–ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor (EGFR), which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID–ABPP is highly complementary to biotin-based enrichment strategies in activity-based protein profiling studies, with PhosID–ABPP providing the advantage of direct ABP-interaction site identification.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin Cancer Cell

SUBMITTER: Wouter van Bergen

LAB HEAD: A.J.R. Heck

PROVIDER: PXD036569 | Pride | 2023-01-10

REPOSITORIES: Pride

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			Action	DRS
	Cell_lysate_Trypsin_IMACenriched_01.raw	Raw
	Cell_lysate_Trypsin_IMACenriched_02.raw	Raw
	Cell_lysate_Trypsin_IMACenriched_03.raw	Raw
	Cell_lysate_Trypsin_IMACenriched_PF-06672131-phosphonate_spectra.pdf	Pdf
	Cell_lysate_Trypsin_IMACenriched_evidence.txt	Txt

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Project description:Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-targets, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy, PhosID-ABPP, on a timsTOF Pro mass spectrometer. This refinement enables dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely PF-06672131 and PF-6422899, facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, while both ABPs showed comparable affinities for the EGFR, PF-06672131 had a broader off-target reactivity profile. In contrast, PF-6422899 exhibited higher affinity for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF-06672131 also effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nanomolar. Additionally, analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on- and off-target engagement in a dose- and site-specific manner.

Dataset Information

SITE-SPECIFIC ACTIVITY-BASED PROTEIN PROFILING USING PHOSPHONATE HANDLES

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