TMT Phosphorylated Protein Proteomics

TMT (Tandem Mass Tag) Phosphoproteomics is an advanced mass spectrometry analysis technique used to quantitatively analyze changes in protein phosphorylation levels. This technique combines the enrichment of phosphorylated peptides, TMT labeling, and high-resolution mass spectrometry analysis, enabling researchers to perform high-throughput, high-precision analysis of protein phosphorylation across multiple samples. It is widely used in biomedical research, especially in the fields of disease mechanisms, drug effects, and cell signal transduction.

 

The core of TMT phosphoproteomics is the use of TMT reagents to isotopically label protein samples. TMT reagents are chemical labels that can bind covalently to the amino terminus of peptides or the side chain amino groups of lysine. Each TMT reagent contains a different isotopic combination, so it can generate labeled peptides of different masses, which can be detected in mass spectrometry analysis. By comparing the TMT signal strength of the same peptides in different samples, changes in protein expression levels can be quantitatively analyzed.

 

Before TMT labeling, it is usually necessary to enrich phosphorylated peptides in the sample through specific enrichment strategies, such as Immobilized Metal Ion Affinity Chromatography (IMAC) or titanium dioxide (TiO2) enrichment. This step is necessary because phosphorylated peptides are relatively low in abundance in protein samples.

 

Analysis Workflow

1. Sample Preparation

First, proteins are extracted and then cut into smaller peptides by enzymatic digestion (usually using trypsin).

 

2. Enrichment of Phosphorylated Peptides

As phosphorylated peptides are usually present in low quantities in protein samples, specific methods (such as immobilized metal ion affinity chromatography) are used to enrich phosphorylated peptides.

 

3. TMT Labeling

Different TMT labels are combined with peptides from different samples. Each TMT label has a unique mass, allowing peptides from different samples to be distinguished during mass spectrometry analysis.

 

4. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Analysis

All labeled samples are mixed and peptides are separated by liquid chromatography, followed by mass spectrometry analysis. In MS/MS analysis, peptides are further broken down, and protein and phosphorylation levels in different samples are quantified by detecting TMT labels.

 

5. Data Analysis

Finally, specialized software is used to analyze the mass spectrometry data to determine protein identity, phosphorylation sites, and quantitative information.

 

The advantage of TMT phosphoproteomics lies in its high-throughput and high sensitivity, which can simultaneously analyze the expression and phosphorylation status of thousands of proteins in multiple samples, providing a powerful tool for biological research.