Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles

Bergen, Wouter van; Hevler, Johannes F.; Wu, Wei; Baggelaar, Marc P.; Heck, Albert J. R.

doi:10.1016/j.mcpro.2022.100455

Cited by 7 publications

(20 citation statements)

References 72 publications

(106 reference statements)

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“…We previously reported PF131’s affinity for Reticulon-4 at cysteine 1101 in intact cells with a concentration of 25 µM [12]. Our current results reveal that RTN4 binding by PF131 can even be detected at a concentration as low as 1 nM, and 10 nM by PF899.…”

Section: Resultssupporting

confidence: 55%

“…This work builds further upon our recent report describing site-specific ABPP using phosphonate handles [12]. To further improve the sensitivity of PhosID-ABPP and facilitate the comparative analysis of multiple ABPs within a complex proteome, we changed to, and optimized the settings on a timsTOF HT mass analyzer for enhanced identification and quantification of ABP-labeled peptides.…”

Section: Resultsmentioning

confidence: 95%

“…We selected the non-specific protease pepsin for the proteolysis of ABP-labeled proteomes, based on previous findings demonstrating that pepsin, unlike trypsin, enables the detection of the primary ABP binding site of PF131 on EGFR [12]. Here again, LC-MS analyses identified the covalent attachment of both ABPs to cysteine 797 (EGFR;C797), within the EGFR active site.…”

Section: Resultsmentioning

confidence: 99%

“…ABPs consist of a 'warhead' to form a covalent bond with target proteins, a recognition element that improves affinity for specific proteins, and a reporter tag to enable visualization or enrichment of targeted proteins [13,[15][16][17][18][19][20][21]. A peptide-centric enrichment approach enables direct liquid chromatography-mass spectrometry (LC-MS) detection of ABP-bound peptides, resulting in a reduction of detected false positive identifications and a site-specific view of the ABP target landscape [12].…”

Section: Introductionmentioning

confidence: 99%

“…Our study employed a peptide-centric ABPP approach using phosphonate-based enrichment tags (PhosID-ABPP) for the two ABPs in a single cellular proteome [12]. Through their distinctive masses, we obtained a detailed view of the exact localization and relative binding affinities of both ABPs over different concentrations, charting their site-specific binding interactions with proteins.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed Drugs

van Bergen,

Fiala,

Heck

et al. 2023

Preprint

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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 HT mass spectrometer. This refinement enables dual 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.

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Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%