TMT/iTRAQ Labeling-Based Quantitative Service

Tandem mass tag (TMT) and isobaric tags for relative and absolute quantification (iTRAQ) are representative isobaric labeling techniques, enabling simultaneous quantitative analysis of up to 16 biological samples. Their core advantages include:

1. High-throughput multiplexed analysis – Allows direct comparison of multiple biological samples (e.g., disease vs. control, time-course studies, multi-dose treatments) in a single experiment, significantly enhancing efficiency.

2. High precision and sensitivity – Mass spectrometry-based signal amplification enables accurate detection of low-abundance protein changes (e.g., inflammatory cytokines or tumor biomarkers in plasma).

3. A bridge to clinical translation – Applicable to complex biological samples, including blood, tissues, and cells, facilitating biomarker discovery, drug target identification, and therapeutic evaluation.

 

As precision medicine advances, TMT/iTRAQ technology has become a pivotal tool for multi-dimensional proteomics research. However, quantitative accuracy depends heavily on optimized experimental design and standardized workflows. With extensive project experience and technological innovation, MtoZ Biolabs offers high-confidence TMT/iTRAQ labeling-based quantitative service, helping clients overcome technical bottlenecks and unlock the full potential of their data.

 



Chen, X. et al. Genomics Proteomics Bioinformatics. 2021.

Figure 1. iTRAQ/TMT Labeling Strategy for Clinical Proteomics

 

Services at MtoZ Biolabs

Our TMT/iTRAQ labeling-based quantitative service cover the entire workflow, including experimental design, sample processing, mass spectrometry analysis, and bioinformatics interpretation, with a focus on:

1. Clinical sample analysis – In-depth profiling of complex biological matrices such as plasma, serum, and tissue lysates.

2. Dynamic process studies – Investigating disease progression and time-resolved drug response in proteomics.

3. Multi-omics integration – Combining transcriptomics and metabolomics to reveal regulatory networks and molecular mechanisms.

 

Whether your research focuses on fundamental biological questions or translational medicine applications, we offer tailor-made solutions to meet your needs.

 

Analysis Workflow

1. Optimized Sample Preparation

• Efficient removal of high-abundance interfering proteins (e.g., albumin and immunoglobulins in plasma).
• Enrichment of low-abundance proteins to improve detection sensitivity.
• Standardized reduction and alkylation procedures to ensure peptide stability and labeling efficiency.

 

2. TMT/iTRAQ Labeling & Fractionation

• Usage of high-purity reagents, with strict control over labeling conditions (pH, temperature, reaction time).
• Strong cation exchange (SCX) or multi-dimensional liquid chromatography (LC) fractionation to reduce sample complexity.

 

3. High-Resolution Mass Spectrometry Analysis

• Equipped with Orbitrap series mass spectrometers, employing DDA (data-dependent acquisition) and MS3 scanning strategies to mitigate quantification bias caused by co-eluting peptides.
• Dynamic exclusion and FAIMS (Field Asymmetric Ion Mobility Spectrometry) to enhance throughput and specificity.

 

4. Data Analysis & Validation

• Database searching and quantitative calculations using industry-standard software.
• Differential protein analysis, combined with KEGG/GO pathway enrichment to identify key targets.

 



Chen, X. et al. Genomics Proteomics Bioinformatics. 2021.

 Figure 2. Overview of a Typical Isobaric Labeling Workflow

 

Why Choose MtoZ Biolabs?

1. Comprehensive Protein Coverage

For plasma samples, we routinely identify >1,200 proteins, a 30% improvement over industry averages.

 

2. Superior Quantitative Accuracy

MS3 scanning and interference correction algorithms reduce ratio compression errors to <15%.

 

3. Stringent Quality Control

Labeling efficiency validation (>98%), background removal with blank controls, and batch quality control (CV <10%) ensure data reliability.

 

4. Flexible Support for Complex Sample Types

Capable of handling low-volume samples (as little as 50 µL of plasma), multiple species (human, mouse, rat, plant), and specialized sample types (e.g., exosomes and cerebrospinal fluid).

 

Applications

• Oncology Research – Identification of key regulatory proteins in the ANGPTL3/VEGF pathway in hepatocellular carcinoma.
• Neurodegenerative Diseases – Quantitative analysis of cerebrospinal fluid in Alzheimer’s disease, detecting early changes in Aβ oligomer-binding proteins.
• Infection & Immunology – Monitoring dynamic changes in plasma inflammatory factors in severe COVID-19 patients, revealing immune storm biomarkers.
• Drug Development – Evaluating the effects of kinase inhibitors on tumor cell signaling networks, accelerating targeted drug discovery.

 

Case Study

1. Optimized TMT-Based Quantitative Cross-Linking Mass Spectrometry Strategy for Large-Scale Interactomic Studies

Cross-linking mass spectrometry (XL-MS) is an effective method for investigating protein-protein interactions (PPI) in complex samples. When combined with TMT labeling, it enables large-scale PPI quantification. However, a robust TMT-based XL-MS quantification strategy has not yet been fully established due to the lack of a benchmarking dataset and comprehensive evaluation of mass spectrometry parameters. This study generates a dual-interactome dataset by spiking TMT-labeled cross-linked Escherichia coli lysate into TMT-labeled cross-linked HEK293T lysate under a defined mixing scheme, allowing systematic assessment of cross-link identification and quantification accuracy across different MS acquisition strategies. The study highlights the need for higher fragmentation energies for TMT-labeled cross-links compared to unlabeled ones and optimizes a stepped HCD-MS2 method (36-42-48 eV), achieving a high number of quantifiable cross-links with improved quantification accuracy. This method is widely applicable for multiplexed quantitative PPI characterization in complex biological systems. TMT/iTRAQ Labeling-Based Quantitative Service integrates TMT/iTRAQ isobaric labeling technology with cross-linking mass spectrometry to enable high-throughput quantitative analysis of protein interaction networks. By employing optimized MS acquisition strategies, it provides in-depth characterization of protein complexes, structural interactions, and dynamic changes. This approach supports interactomics research in various biological systems, facilitating the quantitative profiling of complex protein networks.

 



Ruwolt, M. et al. Anal Chem. 2022.

Figure 3. Evaluation of Cross-Link Identification Using Three SCX Fractions of the TMT-Labeled Two-Interactome Dataset

 

2. iTRAQ-Based Quantitative Proteomic Analysis of the Antimicrobial Mechanism of Lactobionic Acid Against Staphylococcus Aureus

Staphylococcus aureus is a common pathogenic microorganism that causes foodborne diseases. This study employed iTRAQ-based quantitative proteomics to investigate the antibacterial mechanism of lactobionic acid (LBA) against S. aureus and identified 274 differentially expressed proteins upon LBA treatment. The results, combined with ultrastructural observations, suggest that LBA inhibits S. aureus by disrupting cell wall and membrane integrity, regulating ABC transporter expression, affecting cellular energy metabolism, reducing virulence and infection, and decreasing proteins related to stress and starvation responses. These proteomic findings were validated by quantitative real-time PCR, providing new insights into LBA’s inhibitory effects and its potential applications in food and pharmaceutical safety. TMT/iTRAQ Labeling-Based Quantitative Service utilizes TMT/iTRAQ isobaric labeling technology for high-throughput quantitative proteomics, enabling comprehensive analysis of differential protein expression. It supports studies on antimicrobial mechanisms, cellular stress responses, and metabolic regulation, providing detailed insights into protein-level changes under specific treatments. This approach facilitates the investigation of protein function and regulatory pathways in various biological and industrial applications.

 



Cao, J. et al. Food Funct. 2021.

Figure 4. iTRAQ Analysis Reveals Differentially Expressed Proteins after Lactobionic Acid Treatment

 

Deliverables

• Raw mass spectrometry data (RAW files).
• Identified/quantified protein list (including peptide sequences and confidence scores).
• Differential protein expression statistics and visualizations (volcano plots, heatmaps).
• Biological function annotations (pathway enrichment, protein interaction networks).
• Custom analyses – Subcellular localization predictions, combined PTM analysis (phosphorylation/ubiquitination).
• Sample reanalysis & validation – SRM/PRM targeted mass spectrometry or Western blot confirmation.

 

In proteomics research, technical precision determines data success. MtoZ Biolabs adheres to international standards, combining state-of-the-art workflows, an expert technical team, and efficient project delivery to eliminate technical barriers and help researchers focus on their scientific questions. Whether you're new to proteomics or a clinical expert in translational medicine, we strive to be your most trusted partner. Contact MtoZ Biolabs today to start your high-throughput quantitative proteomics journey!