Comparison and Application of Proteomic Technologies

  1. Overview of TMT and iTRAQ Quantitative Proteomics for Peptide Labeling

  2. TMT/iTRAQ Quantitative Proteomics

  3. Label-free DDA Quantitative Proteomics

  4. DIA Quantitative Proteomics

  5. Proteomic Technologies TMT/iTRAQ vs Label Free vs DIA Quantitative Proteomics

  6. How to Choose the Right Proteomics Technique?

  7. Applications of Proteomics

In the ever-evolving field of biotechnology, proteomic technologies stand out as pivotal tools for understanding the complexities of protein dynamics in various biological systems. Proteomics, the comprehensive study and characterization of proteins, is essential in deciphering the molecular mechanisms that underlie various physiological responses and diseases. This article delves into the comparison and applications of advanced proteomic technologies, such as Tandem Mass Tags (TMT) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ). These technologies are reshaping how scientists quantify proteins and understand cellular functions at a molecular level. Through the lens of these powerful tools, we will explore their methodologies, advantages, limitations, and the considerations necessary for choosing the right proteomic technique for your research needs.


Overview of TMT and iTRAQ Quantitative Proteomics for Peptide Labeling

Genes undergo transcription into mRNA, generating multiple alternative splicing forms. During mRNA translation into proteins, they undergo diverse post-transcriptional regulations, processing, modifications, and degradation. Consequently, disparities may arise at the mRNA transcription level, albeit not necessarily at the protein level. Proteins, serving as the principal agents of cellular functions, represent biomacromolecules intricately linked to phenotypes. Proteomics, the discipline dedicated to studying the composition and dynamics of proteins within cells, tissues, or organisms, primarily relies on mass spectrometry for detection. This approach is categorized into non-labeled quantification techniques, such as Label-free and Data-independent acquisition (DIA), and labeled quantification techniques, including TMT and iTRAQ. Additionally, based on the mode of data acquisition, proteomic analysis is classified into Data-dependent acquisition (DDA) and DIA. Notably, Label-free, TMT, and iTRAQ methods predominantly utilize the DDA mode.


TMT/iTRAQ Quantitative Proteomics

TMT and iTRAQ quantitative proteomics are peptide labeling quantitative technologies developed by Thermo Scientific and AB Sciex, respectively. They enable simultaneous protein quantification from up to 16 (18) different samples in a single experiment. This approach involves pooling samples for detection, ensuring excellent parallelism, reducing technical errors, and enhancing detection efficiency. However, it cannot analyze specifically expressed proteins and is unsuitable for samples with significant differences, such as those from diverse species or tissues.


Label-free DDA Quantitative Proteomics

Label-free DDA quantitative proteomics is a method for quantifying proteins without relying on isotopic labeling. In Data Dependent Acquisition (DDA) mode, secondary mass spectrometry selectively fragments and collects information from the top 10/20/40 precursor ions based on their intensity in the primary spectrum. While this approach may introduce randomness and overlook precursor ions with lower abundance. The Label-free DDA quantitative technique analyzes enzymatically digested protein peptides using liquid chromatography to provide relative quantification of corresponding proteins. By employing non-labeled quantification, it identifies differences in the same proteins across multiple samples, offering advantages such as cost-effectiveness, simplicity, and scalability without being constrained by sample numbers. However, optimal results necessitate stable instrument performance and meticulous operation.


DIA Quantitative Proteomics

Data Independent Acquisition (DIA) is a cutting-edge mass spectrometry data acquisition method, hailed as one of the most promising technologies by the esteemed journal 'Nature Methods.' This innovative technique segments the entire mass spectrum scanning range into multiple windows based on mass-to-charge ratio (m/z). Within each window, it systematically fragments and detects all precursor ions, capturing detailed fragment ion information crucial for protein identification and quantification. Compared to the conventional Data Dependent Acquisition (DDA) mode, DIA boasts several advantages, including panoramic scanning capabilities, maximized data utilization, heightened reproducibility, enhanced quantification accuracy, and the provision of traceable data. Consequently, DIA delivers more precise and comprehensive results, rendering it highly suitable for protein detection in extensive sample sets and intricate biological systems.

Proteomic Technologies TMT/iTRAQ vs Label Free vs DIA Quantitative Proteomics


Label Free










Sample size


No limits

No limits

Application range

The same species and tissue

No limits

No limits

Quantitation accuracy





How to Choose the Right Proteomics Technique?

1. TMT Labeling Technology: TMT labeling allows for the simultaneous detection of up to 16 samples in a single run, offering excellent parallelism and accuracy. Therefore, when dealing with fewer than 16 samples, TMT labeling technology is recommended.

2. DIA Technology for Larger Sample Sets: For sample sets exceeding 16 samples, DIA demonstrates greater stability and reproducibility, making it the preferred choice.

3. Consideration for Sample Variability: TMT labeling technology is limited to detecting proteins common across all samples and is not suitable for significantly different samples (such as those from different species or tissues). In contrast, DIA and Label-free quantification are non-labeled methods capable of detecting sample-specific proteins. Therefore, for samples with substantial differences or a focus on sample-specific proteins, DIA or Label-free techniques are recommended.


Applications of Proteomics

Proteomics is a fundamental tool for deciphering life's processes and unraveling the complexities of various diseases. It not only provides insights into the material basis of life activities but also lays the groundwork for understanding disease mechanisms. Below are two publications that showcase the diverse applications and significance of proteomics in biomedical research:

1. Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers (Hoshino A, Kim HS, Bojmar L, et al. 2020)

Abstract Summary: This study delves into the proteomic profile of extracellular vesicles and particles (EVPs) across various human samples, including tissues and bodily fluids. The identified EVP proteins serve as reliable biomarkers for cancer detection and classification, highlighting the diagnostic potential of proteomics in oncology.

2. Proteogenomic Characterization Reveals Therapeutic Vulnerabilities in Lung Adenocarcinoma (Gillette MA, Satpathy S, Cao S, et al. 2020)

Abstract Summary: This comprehensive proteogenomic analysis of lung adenocarcinoma (LUAD) sheds light on therapeutic opportunities through deep-scale proteomics and multi-omics clustering. The study identifies potential therapeutic vulnerabilities associated with key driver mutations, offering valuable insights for targeted therapies in LUAD.

These publications exemplify the broad spectrum of proteomics applications, from cancer biomarker discovery to unveiling therapeutic targets in specific disease contexts. Incorporating proteomics into your research can lead to impactful discoveries and advancements in biomedical sciences.


As the demand for more precise and comprehensive proteomic analyses increases, technologies like TMT and iTRAQ are proving to be invaluable. By providing detailed insights into protein quantification and expression in complex sample sets, these techniques support critical advancements in disease research, biomarker discovery, and therapeutic development. However, the choice of the right proteomic method hinges on understanding the specific requirements and constraints of your experimental setup.


For researchers and institutions looking to leverage the full potential of proteomic technologies, partnering with a leading proteomics service provider like MetwareBio can significantly enhance the outcomes of your scientific investigations. MetwareBio offers innovative metabolomics, lipidomics, and proteomic services that cater to diverse research needs, ensuring high-quality, reliable, and reproducible results. Contact us to explore the possibilities and elevate your research.



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