How to Detect Generalization

Ubiquitin (Ub), unlike the single group added during phosphorylation, methylation, and acetylation modification, is a small protein composed of 76 amino acids, which is widely present in eukaryotic cells and has a highly conserved sequence. Its spatial structure consists of 5 β-folds and 1 α-helix, forming the SSHSSS structure. Ubiquitination refers to the covalent modification of ubiquitin molecules to the ε-amino group of the lysine side chain of the substrate protein under the cascade enzymatic reaction of E1 ubiquitin activating enzyme, E2 ubiquitin binding enzyme, and E3 ubiquitin ligase, or further covalent binding to the lysine residues of the ubiquitin molecule, carrying out the assembly process of different ubiquitin chains.

 

In addition to serving as a signal for protein-specific degradation, recent studies have found that different types of ubiquitin chain signals are widely involved in the cellular localization of target proteins, regulating the stability of proteins, binding with interacting proteins, and transmitting stimulus signals, thereby regulating almost all life processes in the cell, including gene transcription, signal transduction, DNA damage repair, cell cycle regulation, stress response, and even individual immune responses.

 

Analysis Workflow

Mass spectrometry is a common analytical technique in biological research in recent years, and it is currently widely used for the detection of proteins. The technique of detecting ubiquitinated proteins using mass spectrometry includes four steps: sample preparation, enrichment of ubiquitinated peptide segments, biomass spectrometry identification, and data analysis.

 

1. Sample Preparation

The solution or in-gel digestion method is usually used. Use site-specific protease to fully digest the extracted protein mixture.

 

2. Enrichment of Ubiquitinated Peptide Segments

Use an anti-ubiquitin antibody with high affinity, bind it to the ubiquitinated protein, and then combine the ubiquitinated protein with the antibody through immunoprecipitation or affinity chromatography for further analysis.

 

3. Biomass Spectra Identification

After separating the peptide segments by gel electrophoresis or liquid chromatography (LC), use a mass spectrometer for detection. Through mass spectrometry analysis, these ubiquitinated proteins can be qualitatively and quantitatively identified, revealing the position and extent of ubiquitination. The mass spectrometry techniques commonly used for the identification of ubiquitinated proteins mainly include Selective Reaction Monitoring (SRM) and Parallel Reaction Monitoring (PRM).

 

4. Data Analysis

To identify the sequence and modification site of the target protein from the large amount of information of peptide segments or fragment ions obtained from mass spectrometry detection, a specialized protein database search tool is needed. At present, the commonly used Peptide Mass Fingerprint (PMF) database search tools include Mascot, MaxQuant, and Phenyx, and the tools for tandem mass spectrometry database search include Mascot, OMSSA, X! Tandem and pFind.

 

MtoZ Biolabs uses Thermo's latest Obitrap Fusion Lumos mass spectrometer combined with Nano-LC to provide a one-stop service for protein ubiquitination detection for researchers. Just tell us your needs and send your samples, MtoZ Biolabs is responsible for all subsequent projects, including protein extraction, protein digestion, ubiquitinated peptide segment enrichment, peptide separation, mass spectrometry analysis, raw mass spectrometry data analysis, bioinformatics analysis, and provides you with detailed bilingual technical reports in both Chinese and English. Feel free to consult for free.