Abstract
Targeting E3 ubiquitin ligases with chemical probes remains a major challenge, despite their central roles in cellular regulation and disease. Constitutive photomorphogenic 1 (COP1) is an E3 ligase whose complex, adaptor-dependent biology has made its selective targeting difficult. Here, we develop a peptide-based affinity selection mass spectrometry (Pep-AS-MS) platform to discover new peptide based COP1 binders. Leveraging a mass spectrometry compatible library design, fluorescence-guided selection optimisation, and an open-source software driven target-decoy database strategy for peptide identification and quantification, we enable hit ranking from highly complex combinatorial libraries. Using this approach, we identify and validate six previously unreported COP1-binding peptides, including ligands with binding potencies comparable to the native TRIB1 motif. This work establishes a quantitative and accessible Pep-AS-MS framework for ligand discovery against challenging protein–protein interaction targets and provides new chemical tools to interrogate COP1 biology. Screening of COP1 E3 ligase with pooled, peptide-based combinatorial libraries and affinity selection mass spectrometry revealed new peptide binder sequences for further development as cellular probes for studying COP1 biology.