TMT Based Proteomics Service

What Is TMT?

The TMT, or Tandem Mass Tag Reagents, is a well-established stable isotope labeling reagent developed by Thermo Fisher Scientific. TMT quantification belongs to the isobaric isotope labeling protein quantitative method, and remains the most widely adopted approach among quantitative methods. The mass-tagging reagent within a set consists of an amine-reactive NHS-ester group (Amine Reactive Group), a spacer arm (balance group), and a mass reporter group. Based on the principle of isotope, peptides from different sources are labeled by linking different isotope labels to N- terminus of a peptide or to a lysine residue, particularly under pH 8.5. In a specific combination kit, the three groups ensure a constant total molecular weight through different numbers and combinations of 13C and 15N isotopes at different locations, resulting in monoisotopic mass differences of 6 mDa in the reporter and these differences can be accurately quantified using high resolution mass spectrometry (MS), so as to realize the differentiation of peptide sources after labeling peptide segments.

Figure 1. The general chemical structure formula of the TMT reagent.

Importantly, up to now, TMT kits include TMT⁶-plex, TMT¹⁰-plex, TMT¹¹-plex, TMTPro¹⁶-plex, and TMTPro¹⁸-plex. It can be used to label up to 18 different samples. For each sample, a unique reporter mass (i.e., 126-135Da) in the low mass region of the MS/MS spectrum is used to measure relative protein expression levels during peptide fragmentation. Which offers multiplexing capabilities for relative quantitative proteomics analysis. This TMT label technique has been widely adopted in studies focusing on differential expression proteome analysis and the discovery of protein biomarkers for diseases.

Table 1. Modification masses of the Thermo Scientific TMT Label Reagents.

* HCD (higher-energy collision dissociation) is a collisional fragmentation method that generates ten unique reporter ions from 126 to 131Da.

** ETD (electron transfer dissociation) is a non-ergodic fragmentation method that generates six unique reporter ions from 114 to 119Da.

How Does TMT Labeling Work?

TMT proteomics workflow

Figure 3. Workflow of TMT labeling proteomics. (Yuste-Montalvo, Alma et al. Frontiers in immunology, 2021)

In MS1 spectrum, the same peptide segment from different labeled samples showed a peak, due to the same peptide segment in different samples labeled by TMT reagent showed the same mass-to-charge ratio.

In MS2, the chemical bond between the reporter group, the balance group and the reaction group are broken, and the TMT reporter group and the balance group are released. The neutral loss of the balance group occurs, and the reporting groups are detected and recorded by MS. TMT reported ion peaks are generated in the low-mass region 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. The qualitative and relative quantitative information of proteins can be obtained from these raw data through database retrieval.

Technical Platforms

Instrument picture from the official website

Thermo Q Exactive^TM series

AB Sciex 6500+

Thermo Orbitrap Fusion Lumos

Bruker timsTOF Pro

Our TMT Based Proteomics Service

Our TMT-based proteomics service enables high-throughput, accurate protein quantification across multiple samples in a single experiment. The workflow is as follows:

Sample Labeling: Different TMT labeling reagents are reacted with individual samples, ensuring all peptide segments within a single sample are tagged with a specific label.

Peptide Pooling: After labeling, equal amounts of peptides from all samples are mixed.

Separation: The mixed peptides are separated using offline reversed-phase liquid chromatography (RPLC).

Mass Spectrometry Analysis: During MS detection, the primary parent ions of identical peptides across different samples appear as a single peak due to their identical total molecular weight, representing a combined signal from all samples.

Selected precursor ions are fragmented, and the reporter ions from different samples are released and detected, enabling precise quantification.

Figure 2. Procedure schematic for quantitative proteomics using TMT ¹⁰-plex Label Reagents.

In addition to TMT-based proteomics, our services also include other protein quantification solutions, such as iTRAQ (Isobaric Tags for Relative and Absolute Quantification). Like TMT, iTRAQ utilizes isobaric labeling for multiplex quantification of proteins and peptides but is optimized for slightly different experimental needs, offering flexibility for diverse research goals.

TMT Labeling Quantitative Proteomic Analysis

TMT Based Proteomics Case Study

Reference

1. Shibata, Hirofumi et al. "A Non-targeted Proteomics Newborn Screening Platform for Inborn Errors of Immunity." Journal of clinical immunology vol. 45,1 33. 25 Oct. 2024.