biostudies-literatureSaidjalolov SSwiss National Science FoundationNCCR CatalysisEuropean Research CouncilUniversit? de Gen?ve3288-3298https://www.ebi.ac.uk/biostudies/studies/S-EPMC12308380biostudies-literatureUnknown5(7)Although facilitated cellular entry of substrates with thiol-reactive motifs has been observed for decades, this so-called thiol-mediated uptake (TMU) remains poorly understood. We have proposed a mechanism of entry involving cellular proteins that form reversible dynamic covalent bonds with thiol-reactive cascade exchangers (CAXs), which is challenging to prove because the substrate-protein bond is transient and constantly shifting. Thus, with conventional proteomics analysis of TMU, continuing exchange during processing should result in labeling of the inert binders rather than the best exchangers, that is, repressors and intracellular targets, instead of the enablers of TMU. Any static covalent bonding to a binding site will also perturb the molecular relay network of interest. The emerJACS AuPhotocatalytic Microenvironment Proteomics of Thiol-Mediated Uptake.PMC12308380200020_18840651NF40-182895TMAG-2_209190209190200020_204175Pologne BSaidjalolov SRose NGajic JMatile SRenno GMercier VWu YMoreau DWinssinger NSakai NfalsePhotocatalytic Microenvironment Proteomics of Thiol-Mediated Uptake.Although facilitated cellular entry of substrates with thiol-reactive motifs has been observed for decades, this so-called thiol-mediated uptake (TMU) remains poorly understood. We have proposed a mechanism of entry involving cellular proteins that form reversible dynamic covalent bonds with thiol-reactive cascade exchangers (CAXs), which is challenging to prove because the substrate-protein bond is transient and constantly shifting. Thus, with conventional proteomics analysis of TMU, continuing exchange during processing should result in labeling of the inert binders rather than the best exchangers, that is, repressors and intracellular targets, instead of the enablers of TMU. Any static covalent bonding to a binding site will also perturb the molecular relay network of interest. The emer2025-01-01T00:00:00Z2025 Jul2026-03-27T15:56:47.856Z2025-08-27T03:07:43.426ZS-EPMC123083804074702010.1021/jacsau.5c00432