Detailed Explanation of TMT Labeling Quantitative Mass Spectrometry Workflow

As life sciences research advances towards more refined and large-scale system levels, quantitative proteomics becomes a crucial tool for deciphering the dynamic changes in biological systems. Particularly in research areas such as tumor heterogeneity, drug mechanism exploration, and disease biomarker screening, mass spectrometry-based quantitative methods show great potential for application. Among them, TMT (Tandem Mass Tag) labeling technology, with its high throughput, low batch variation, and high quantitative accuracy, has become the preferred method for multi-condition comparative experiments.

I. What is TMT Labeling? A Brief Overview of the Principle

TMT is a chemical tagging technique based on isotopic coding, which achieves relative quantitative analysis of multiple samples by labeling peptides from different samples with reagents that have equal mass and identical structure but release different reporter ions upon fragmentation.

Each TMT reagent consists of three key structural units:

• Reporter group: Used for MS2 or MS3 quantification (m/z 126–131, expandable to TMTpro 16-plex or 18-plex)

• Balancer group: Adjusts the overall molecular mass of the tag to ensure equal mass of each reagent before labeling

• Reactive group: Reacts with the amino group of peptide N-terminus or lysine side chain for covalent labeling

Since all samples are mixed and jointly enter the LC-MS/MS process after labeling, batch variation is significantly reduced, and experimental repeatability and quantitative consistency are greatly enhanced.

II. Standard Workflow of TMT Labeling Quantitative Mass Spectrometry

1. Protein Extraction and Quantification

The experimental workflow begins with protein extraction from samples, and an appropriate lysis method (such as RIPA, SDS, urea, etc.) is chosen based on sample type (cells, tissues, serum/plasma, etc.). The key requirement is to ensure thorough dissolution and good integrity of the proteins.

Subsequently, protein quantification is performed using BCA or Bradford methods to ensure consistent input amounts for each sample (typically recommended ≥ 100 μg).

2. Protein Digestion (Digestion Efficiency Directly Affects Quantitative Quality)

Trypsin digestion is the mainstream approach, often paired with a Lys-C dual enzyme system to enhance cleavage efficiency, especially suitable for high-salt or highly denatured samples. The digestion step typically includes the following procedures:

• Protein reduction (DTT)

• Alkylation (IAA)

• Overnight digestion (37°C, enzyme:substrate=1:50)

This step yields a peptide mixture, which is subsequently used for TMT labeling.

3. TMT Labeling Reaction

Each group of samples is labeled with different TMT reagents, with reaction conditions including:

• Solution buffer environment: Typically using amine-free buffers (such as TEAB)

• Reaction time: 60 minutes at room temperature

• Stopping the reaction: Adding ammonium hydroxide or amino acids to neutralize excess reagents

Labeling efficiency should be >95%, and Biotech Pac sets quality control points during the process, using small-scale samples to verify complete labeling.

4. Sample Mixing and Fractionation

After labeling, different samples are mixed in equal amounts to obtain a unified composite sample mixture. At this time, the sample complexity is high, requiring further fractionation through high-pH reverse-phase chromatography to enhance mass spectrometry coverage.

Common methods include:

• High-pH reverse-phase chromatography

• Ion exchange (SCX)

• Electrophoresis (SDS-PAGE, OFFGEL)

The number of fractions generally ranges from 8 to 20, depending on sample complexity and research depth requirements.

5. LC-MS/MS Analysis (High-Resolution Mass Spectrometry Platform)

Entering the mass spectrometry analysis stage, Biotech Pac uses high-end platforms such as Orbitrap Exploris 480 / Fusion Lumos / timsTOF, combining nano liquid chromatography (nanoLC) to achieve quantification at the MS2 or MS3 level.

Key parameter settings include:

• Electrospray ionization (ESI)

• Positive ion mode

• DDA mode or DIA+DDA mixed strategy

• TMT quantification mode (MS2 or SPS-MS3)

Among them, the SPS-MS3 mode can effectively reduce the ratio compression issue caused by co-fragmentation, enhancing quantitative accuracy.

6. Data Analysis and Bioinformatics Interpretation

The raw mass spectrometry data is parsed by software such as:

• Proteome Discoverer (Thermo platform)

• MaxQuant (compatible with multiple platforms)

• Spectronaut (DIA compatible)

Matching databases (Uniprot/RefSeq), filtering reliable peptides (FDR<1%), outputting protein expression data matrix.

Then proceed to differential protein screening, GO/KEGG enrichment analysis, clustering analysis, network analysis (PPI), visualization mapping, and other stages.

III. Advantages of Biotech Pac's TMT Service System

In the direction of TMT quantitative proteomics, Biotech Pac relies on its self-built high-throughput mass spectrometry platform and mature sample processing system to provide customers with end-to-end one-stop solutions:

1. Rich experience in sample pre-processing, adaptable to various complex types (FFPE, plasma, micro tissue);

2. Flexible support for TMT 6/10/11/16/18 plex labeling;

3. In-depth data analysis, including biological pathways, disease enrichment, and target prediction;

4. Strict quality control: monitoring labeling efficiency, protein coverage, and technical reproducibility throughout the process;

5. Can be integrated with metabolomics, transcriptomics, and other omics for multi-omics joint analysis.

TMT-based mass spectrometry has become a core technology for exploring changes in protein expression, particularly suitable for multi-group, multi-replicate, and comparative studies with clear research designs. Whether in basic research or clinical translation, accurate and reproducible quantitative results are the foundation of data credibility. Baitai Parker Biotechnology will continue to optimize the TMT quantitative mass spectrometry platform to deliver data with high standards, provide high-quality support for scientific research, and offer solid support for life sciences research.