Tandem Mass Tag Quantitative Proteomics

Tandem Mass Tags (TMT) quantitative proteomics is an advanced mass spectrometry technique used to simultaneously quantify the expression of proteins in multiple samples. This method is particularly suitable for the quantitative comparison of proteins in complex biological samples, and is of great significance for disease mechanism research, biomarker discovery, and analysis of drug action mechanisms.

 

The Definition of TMT

TMT is a mature stable isotope labeling reagent developed by Thermo Fisher Scientific. TMT quantification belongs to the isotope-labeling quantification method, which is still the most widely used method in quantification methods. The mass tag reagent in a set consists of an amine-reactive NHS-ester group (amine-reactive group), a spacer arm (balancing group), and a mass reporting group. Based on the principle of isotopes, different isotopic labels are connected to the N-terminus or lysine residues of peptides by labeling peptides from different sources, especially at pH 8.5. In a specific combination reagent box, three groups ensure a constant total molecular weight through different numbers and combinations of 13C and 15N isotopes in different positions, resulting in a single isotopic mass difference of 6 mDa in the reporting gene. These differential masses can be quantified accurately using high-resolution mass spectrometry (MS), thus distinguishing the source of the labeled peptide segments.

 

Importantly, TMT reagent kits to date include TMT 6-plex, TMT 10-plex, TMT 11-plex, TMTPro 16-plex, and TMTPro 18-plex. It can be used to label up to 18 different samples. For each sample, the unique reporting mass in the low mass region of the MS/MS spectrum (i.e., 126-135Da) is used to measure the relative protein expression levels during the peptide fragmentation process. It provides multiplex functionality for relative quantitative proteomics analysis. This TMT labeling technique has been widely used in the study of differential protein group analysis and disease protein biomarker discovery.

 

Principle of TMT-Based Proteomics

In the MS1 spectrum, peaks appear for the same peptide segment in different labeled samples due to the same mass-to-charge ratio exhibited by the TMT-labeled different samples.

 

In MS2, the chemical bonds between the reporting group, balancing group, and reactive group break, and the TMT reporting group and balancing group are released. Balanced group neutral loss occurs, and the MS records and reports the group. The ion peaks of TMT reporting occur in the low mass area of the MS2 spectrum, and their intensity reflects the relative expression information of the peptide in different samples. In addition, the mass-to-charge ratio of the peptide fragment ion peak in MS2 reflects the sequence information of the peptide. By database retrieval, qualitative and relative quantitative information of proteins can be obtained from these raw data.

 

Advantages of TMT Technology

1. Breadth

TMT reagent kits are compatible with samples from various sources, including cells, animal and plant tissues, bacteria, blood, subcellular protein fragments, etc.

 

2. High Adaptability

It's also applicable for PTMs and IP-MS analysis.

 

3. High Specificity

The efficiency of TMT labeling is over 99%.