biostudies-literatureUnknown24(2)Huppelschoten YSmall ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83 isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screeneChembiochem : a European journal of chemical biologye202200601https://www.ebi.ac.uk/biostudies/studies/S-EPMC10107784biostudies-literatureIn-Plate Chemical Synthesis of Isopeptide-Linked SUMOylated Peptide Fluorescence Polarization Reagents for High-Throughput Screening of SENP Preferences.PMC10107784Vertegaal ACOvan der Heden van Noort GJHuppelschoten YOvaa HMukhopadhyay RBuchardt JNielsen TEfalseIn-Plate Chemical Synthesis of Isopeptide-Linked SUMOylated Peptide Fluorescence Polarization Reagents for High-Throughput Screening of SENP Preferences.Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83 isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screene2023-01-01T00:00:00Z2023 Jan2025-04-26T04:59:38.971Z2025-04-06T11:20:40.221ZS-EPMC101077843637760010.1002/cbic.202200601