How to Determine Ubiquitination Sites

Ubiquitination is a post-translational modification process in which a small protein called ubiquitin is attached to other proteins. Determining ubiquitination sites is a complex process that can be conducted largely through the following methods:

 

1. Bioinformatics Prediction

Specialized software and databases are used to predict ubiquitination sites. Tools such as UbPred and Ubpred can be used for ubiquitination site prediction. These tools are often based on known ubiquitination sites and their sequence characteristics for predictions.

 

2. Experimental Validation

(1) Mass Spectrometry Analysis

Mass Spectrometry (MS) technology is used to identify ubiquitination sites in protein samples. In this process, specific enzymes are usually used to cut proteins, followed by analysis of their peptides to determine the specific location of ubiquitination.

 

(2) Immunoprecipitation

Specific antibodies (e.g., anti-ubiquitin antibodies) are used for immunoprecipitation experiments to enrich ubiquitinated proteins. These are then identified by Western blot or mass spectrometry analysis.

 

(3) Mutation Analysis

Amino acids at predicted ubiquitination sites are mutated, usually by mutating lysine (K) to other amino acids. The impact of these mutations on protein ubiquitination levels is then observed to verify whether these sites are indeed involved in ubiquitination.