Parallel Reaction Monitoring (PRM) Detection of Ubiquitination

Parallel Reaction Monitoring (PRM) is a high-resolution mass spectrometry technique commonly used for targeted proteomics research. It can be used to detect and quantify specific proteins and protein modifications, such as ubiquitination.

 

Ubiquitination is a common form of protein post-translational modification in cells, where proteins modified in this way are linked to one or more ubiquitin proteins. Ubiquitination can determine the stability, location, activity, and cell processes of proteins.

 

Analysis Workflow

1. Sample Preparation

First, a sample containing ubiquitinated proteins needs to be prepared. This might involve cell culture, protein extraction, enrichment of ubiquitination sites (possibly through techniques like immunoprecipitation), and other steps.

 

2. Peptide Preparation

Protein mixtures are digested into peptides by enzymes (such as trypsin), allowing each peptide to be analysed by mass spectrometry rather than the entire protein.

 

3. Mass Spectrometry Analysis

After the peptides are separated by liquid chromatography (LC), they are detected by a mass spectrometer. In PRM analysis, researchers pre-set a series of specific peptides or specific fragments of peptides to be targeted. These targets are selected based on the expected mass/charge ratio of ubiquitinated proteins or specific ubiquitination sites.

 

4. Data Collection

The mass spectrometer collects data on all the fragments of all pre-set peptides. This is a major difference between PRM and traditional SRM (Selected Reaction Monitoring) techniques. SRM monitors only a few pre-selected fragments, while PRM monitors all possible fragments, providing more comprehensive information.

 

5. Data Analysis

The ubiquitinated peptides present in the sample are identified and quantitatively analysed by comparing the experimental data with the theoretical mass and fragmentation patterns of known ubiquitinated peptides.

 

The advantage of PRM is its high accuracy and sensitivity, making it very suitable for detecting low-abundance protein modifications like ubiquitination. Additionally, since it collects all possible fragments of target peptides, it can provide detailed information about ubiquitination sites and types of ubiquitination (like mono-ubiquitination or multi-ubiquitin chains). However, PRM requires a high-resolution mass spectrometer and typically relies on a known target peptide database, which means it might not be suitable for exploring unknown ubiquitination sites.