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A membrane-permeable and IMAC-enrichable crosslinking reagent for advanced in vivo crosslinking mass spectrometry

ABSTRACT: We developed a novel membrane-permeable and IMAC-enrichable cross-linker tBu-PhoX. Applying our optimized in vivo XL-MS pipeline, we revealed PPI landscape from intact human cells.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Heart, Liver, Cell Culture

SUBMITTER: Pin-Lian Jiang

LAB HEAD: Fan Liu

PROVIDER: PXD029033 | Pride | 2024-02-09

REPOSITORIES: Pride

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			Action	DRS
	Human_uniprot_2020_reviewed_2021.09.28.filtered_cross-linked_peptides.csv	Csv
	L1_20210721_JPL_mito_tphoX_LIMAC_CS2-8_1.raw	Raw
	L1_20210721_JPL_mito_tphoX_LIMAC_CS2-8_2.raw	Raw
	L1_20210721_JPL_mito_tphoX_LIMAC_CS3-8_1.raw	Raw
	L1_20210721_JPL_mito_tphoX_LIMAC_CS3-8_2.raw	Raw

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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.

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A membrane-permeable and IMAC-enrichable crosslinking reagent for advanced in vivo crosslinking mass spectrometry

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