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Updated: Aug 5, 2026

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
Published on: March 23, 2020
Synthesis of Linkage-Specific Polyubiquitin Probes Using Unnatural Amino Acids
Matthew T Buhl1, T Ashton Cropp2
1Department of Chemistry, Virginia Commonwealth University, Richmond, VA, 23284, USA.
Abstract:
Activity-based probes based on polyubiquitin chains are powerful tools for studying ubiquitin-conjugating pathways and the substrate specificity of deubiquitinase (DUB) enzymes. These probes can be synthesized through a genetically encoded unnatural amino acid (UAA), Nε-L-thiaprolyl-L-lysine (ThzK). This chapter details the chemical synthesis of ThzK and preparation of diUb probes, leveraging ThzK to enable the production of site-specifically installed isopeptide bonds. The resulting probes facilitate mechanistic studies of ubiquitination and deubiquitination specificity and have applications in understanding diseases associated with ubiquitination dysfunction.
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