Ubiquitin Labeling Free Mass Spectrometry Analysis

Ubiquitination is a key post-translational modification process that involves covalently attaching small ubiquitin proteins to target proteins. This modification can regulate the stability, activity, and localization of target proteins. Mass spectrometry of ubiquitination can identify which proteins are ubiquitinated and the exact sites of ubiquitination. Label-free mass spectrometry is a method that does not depend on isotope or fluorescence labeling. It directly estimates the abundance of proteins by measuring the ion flow of each peptide segment.

 

Analysis Workflow

1. Sample Preparation

(1) Extract proteins from cells or tissues.

(2) Use an enzyme such as Trypsin to digest the proteins, producing peptides.

 

2. Enrich Ubiquitinated Peptides

(1) Because ubiquitinated peptides may be of lower abundance in the overall sample, enrichment is usually necessary.

(2) Use ubiquitin affinity antibodies or other enrichment strategies (such as K-ε-GG acyl peptide enrichment).

 

3. LC-MS/MS Analysis

(1) Use liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to analyze the sample.

(2) The characteristic of ubiquitination is to produce a peptide with a GG modification (from the last two amino acids of the C-terminus of ubiquitin).

 

4. Data Analysis

(1) Use mass spectrometry analysis software (such as MaxQuant, Skyline, etc.) to process and analyze the data.

(2) Estimate the degree of ubiquitination based on the abundance of ubiquitinated peptides.

(3) Identify ubiquitination sites.

 

5. Verification

Verify the results of mass spectrometry analysis by other methods (such as Western Blot or immunoprecipitation).

 

6. Biological Interpretation

Interpret the biological significance and functional impact of ubiquitination based on existing literature and databases.

 

Label-free mass spectrometry, with its no pre-labeling requirement, high throughput, high sensitivity, wide dynamic range, and accurate quantification ability, plays a crucial role in ubiquitinated protein research. It lays the foundation for a deeper understanding of its biological functions and regulatory mechanisms.