Ubiquitination Detection Methods

Ubiquitination is a multi-functional post-translational modification (PTM) that regulates various fundamental properties of protein substrates, including stability, activity, and localization. Ubiquitination involves linking ubiquitin protein (Ub) to substrate proteins through covalent bonds. Ub can covalently link to one or more accessible modified residues of the substrate, forming mono-ubiquitination and multi-mono-ubiquitination, respectively.

 

Characterizing protein ubiquitination is a formidable challenge. Firstly, under normal physiological conditions, the stoichiometry of protein ubiquitination is very low, which increases the difficulty of identifying ubiquitinated substrates. Secondly, Ub can modify one or more lysine residues on the substrate at the same time, which significantly increases the difficulty of locating ubiquitination sites using traditional methods. Thirdly, Ub itself can serve as a substrate, leading to complexity of Ub chains, with varying lengths, connections, and overall structures. In this issue, we summarize the methods or strategies used to analyze ubiquitinated proteins, ubiquitination sites of proteins, and Ub connections and their chain structures.

 

Western Blot

Western blot is a traditional biochemical detection method. By using specific antibodies against ubiquitin and the target protein, the level of ubiquitination of a specific protein can be detected. Ubiquitinated proteins usually appear as bands higher in molecular weight than unmodified proteins, sometimes forming a "ladder" pattern, reflecting the formation of multi-ubiquitin chains.

 

Immunoprecipitation (IP)

This utilizes antibodies against ubiquitin or specific substrate proteins to enrich ubiquitinated proteins, which are then detected by western blot. This method can be used to study the ubiquitination status and ubiquitination sites of specific proteins.

 

Mass Spectrometry (MS)

Mass spectrometry is a powerful technique. With the advancement of MS, researchers are increasingly shifting their focus to proteomic analysis of ubiquitination based on MS. Through mass spectrometry analysis, ubiquitinated peptides and their specific modification sites can be accurately identified, providing detailed information for a deeper understanding of ubiquitination function. MS-based analysis has become the most powerful tool for accurately identifying PTMs. The enrichment methods developed in combination with advanced MS can identify tens of thousands of ubiquitination sites corresponding to thousands of ubiquitinated proteins.

 

Enzyme-Linked Immunosorbent Assay (ELISA)

Using specific antibodies against ubiquitin and the target protein, ELISA can quantitatively detect the level of ubiquitinated proteins. This method is suitable for rapid screening or quantitative analysis.

 

Each method has its advantages and limitations, and researchers choose the most appropriate method based on research objectives and sample types. For example, mass spectrometry analysis provides the most direct information on ubiquitination sites, while Western blotting and immunoprecipitation are more suitable for detecting the ubiquitination status of specific proteins.