TMT Based Proteomics Service

What is TMT?

The TMT, or Tandem Mass Tag, is a well-established stable isotope labeling reagent developed by Thermo Fisher Scientific. TMT quantification belongs to the isobaric isotope labeling 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.

Importantly, up to now, TMT kits include TMT⁶-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, 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.

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

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.

The principle of TMT based proteomics

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.

Our TMT based proteomics service

The basic process is as follows: Different TMT labeling reagents and different samples are reacted and labeled, so that all peptide segments in one sample are labeled with a specific labeled reagent. After all samples were labeled, equal amounts of peptides were mixed, then separated by offline reversed-phase liquid chromatography (RPLC) and detected by high resolution MS. In MS detection, the primary parent ions of the same peptide segment from different samples appear as a peak due to their identical total molecular weight. This phenomenon can be considered as the summation of signals from multiple samples of the peptide segment. When the peptide precursor ion is selected and fragmented, the reporter groups of the labeled reagents from different samples are second fragmented and form free ions, which are detected by MS and finally quantified.

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

In Creative Proteomics, we are professional enough to provide a feasible experimental scheme tailored to your specific requirements to complete the TMT quantitative proteomics research. Our experienced proteomics research team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensuring high-quality and reliable results for quantitative proteomics analysis.

Technologies platform

1. Professional detection and analysis capability: Experienced technical team, strict and skillful techniques.
2. Breadth: TMT kits demonstrate compatibility with samples derived from a wide range of sources, including cells, animal and plant tissues, bacteria, blood, subcellular protein fraction, etc.
3. High adaptability: The quantitative technology is also applicable for the analysis of PTMs and IP-MS.
4. High stability and reproducible: Reducing technical variation in the experimental workflow, obtain consistent and reproducible inter- and intra- assay results for data analysis.
5. High specificity: TMT labeling efficiencies of > 99%.
6. High resolution and sensitivity: Triple TOF 5600, Q-Exactive, Q-Exactive HF, Orbitrap Fusion™ Tribrid™.

Samples Requirement

We can accept a variety of samples, including but not limited to:

Tissue: animal tissue > 50 mg;

fresh plant > 100 mg;

Cell: suspension cell > 1 x 10⁷;

adherent cell > 1 x 10⁷;

microorganism > 50 mg or 2 x 10⁷ cells;

Body fluid: serum/plasma > 100 μL;

Protein: Total protein >300 μg and concentration >1 μg/μL.

Note: In order to ensure the test results, please inform the buffer components if you give us protein lysate, whether it contains thiourea, SDS, or strong ion salts. In addition, the sample should not contain components such as nucleic acids, lipids, and polysaccharides, which will affect the separation effect.

TMT Labeling Quantitative Proteomic Analysis

Results Delivery

1. Detailed report, including experiment procedures, parameters, etc.;
2. Raw data and data analysis results

How to place an order

Please feel free to contact us via email for a comprehensive discussion regarding your specific requirements. Our experienced experts will provide a feasible experimental scheme tailored to your specific requirements, ensuring high-quality results for protein analysis. Besides, customer service representatives are available round the clock, seven days a week.

Reference

1. Zecha J, Satpathy S, Kanashova T, et al. TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach. Molecular & Cellular Proteomics. 2019 Jul;18(7):1468-1478.

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans

Journal: Nature Communications

Published: 2023

Main Technology:  TMT-based quantitative proteomic, ELISA, Area Under the Curve (AUC) Receiver Operating Characteristic (ROC) analysis

Background: Drug-induced liver injury (DILI) is a major clinical problem associated with significant morbidity and mortality. Most cases of DILI recover after early detection of causative medication and its discontinuation. Early detection and diagnosis of DILI is a major challenge as current biomarkers do not distinguish DILI from acute liver injury due to other etiologies. DILI and its distinction from other liver diseases are significant challenges in drug development and clinical practice.

Methods: This study utilized mass spectrometry (MS) with higher-order multiplexing via an isobaric labeling strategy to simultaneously identify and quantify serum proteins in multiple cohorts as sensitive and specific biomarkers for early detection and diagnosis of DILI. We combined Tandem Mass Tag (TMT) based reporter methodology with MS instrumentation capable of providing quantitative accuracy using synchronous precursor MS3 analysis that eliminates interference. Then we developed a targeted MS assay to assess the performance characteristics of the selected candidate biomarkers in a second longitudinal confirmatory cohort. Finally, the performance characteristics of top biomarkers were tested in a third, multicenter, prospective cohort.

Results: 1) During the discovery stage, 2323 proteins were identified in a cohort comprising patients with DILI; 2) The comparative analysis of DO vs HV，DO vs DF，NDO vs DO，NDO vs HV, a total of 89 proteins showed significant differential expression in respective pairwise comparisons and 51 proteins showed significant differences between at least two groups; 3) 13 proteins were selected as candidate biomarkers; 4) A positive biomarker panel (FBP1 + GSTA1 + LECT2) had the best performance, and the model had the highest specificity in identifying patients with DILI.

Conclusions: Through a large multicentre case-control study, the researchers used proteomic techniques and subsequent bioinformatics analysis to reveal that the prediction model based on FBP1+GSTA1+LECT2 can identify DILI and non-DILI liver disease, highlighting the important role of proteomic techniques in the clinical study of DILI.

Fig. 1: Schematic overview of the strategy for discovery, confirmation, and replication of DILI candidate biomarkers.

Creative Proteomics has been dedicated to the field of life sciences and life technology, pioneering multi-omics integration experiments and analysis based on proteomics and metabolomics in the early stages. After years of development and accumulation, the company has established proteinomics technology platforms, including iTRAQ/TMT, DIA, PRM, modified proteomics, as well as comprehensive metabolomics technology platforms, including full-spectrum metabolomics, targeted metabolomics, and lipidomics. Corresponding data integration and analysis platforms have also been established, along with a scientifically sound service workflow and precise operating standards.