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Updated: May 28, 2026
11:54
Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
Published on: March 23, 2020
Analysis of protein ubiquitination
Jeffrey D Laney1, Mark Hochstrasser2
1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island.
Current Protocols in Protein Science
|November 3, 2011
Summary
This study details methods for detecting protein ubiquitination activity, a key step in cellular signaling. These techniques help researchers identify enzymes involved in ubiquitin transfer across various cell types.
Area of Science:
- Biochemistry and Molecular Biology
- Cellular Signaling
Background:
- Protein ubiquitination is a critical post-translational modification regulating numerous cellular processes.
- Enzymes mediate the attachment of ubiquitin (Ub) to target proteins, involving substrate recognition and Ub transfer.
Purpose of the Study:
- To describe and validate methods for assessing protein Ub-transferring activity.
- To provide researchers with practical techniques for studying ubiquitination pathways.
Main Methods:
- Immunoblotting of immunoprecipitated proteins.
- Affinity purification utilizing His-tagged ubiquitin.
- Assaying auto-ubiquitination of E3 ligases.
- In vitro and in E. coli ubiquitination assays using model substrates and Ub-ligation enzymes.
Main Results:
- The described methods effectively determine Ub-transferring activity.
- Techniques are applicable to various eukaryotic cells, with specific protocols for yeast and mammalian cells.
Conclusions:
- A suite of reliable methods is presented for the characterization of ubiquitination enzymes.
- These assays facilitate the investigation of ubiquitination in diverse biological contexts.
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