Working Mechanism and Advantages of the iTRAQ Labeling Technique
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Sample quality assessment and standardized processing.
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Optimization and quality control of isotope labeling.
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Data acquisition using Orbitrap high-resolution mass spectrometry.
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Bioinformatics support including differential protein screening, GO/KEGG pathway enrichment, and PPI network analyses.
High-throughput quantitative analysis across multiple biological samples represents a central objective in proteomics research. With advances in mass spectrometry instrumentation, the iTRAQ labeling technique has gained widespread application in studies of disease mechanisms, drug mechanism-of-action, and biomarker discovery due to its sensitivity, quantitative accuracy, and capacity for multiplexed sample processing.
Working Mechanism of the iTRAQ Labeling Technique
1. Principle
iTRAQ is an isobaric isotope labeling-based quantitative proteomics technique. Its quantitative principle relies on chemically tagging peptides derived from different biological samples with isobaric labels of equal mass. Upon MS/MS fragmentation, these labels generate sample-specific reporter ions that enable relative quantification across multiple samples within a single analysis.
2. Workflow
(1) Protein extraction and digestion: Proteins from multiple samples are extracted and enzymatically digested into peptides using trypsin.
(2) iTRAQ reagent labeling: Each sample is individually labeled with iTRAQ reagents bearing distinct reporter ion numbers (e.g., 114, 115, 116; supporting up to 8- or 10-plex analysis).
(3) Sample mixing: The labeled peptide samples are subsequently combined into a single mixture for downstream mass spectrometric analysis.
(4) LC-MS/MS analysis: At the MS1 (precursor) level, peptides remain indistinguishable due to identical mass tags. During MS/MS fragmentation, reporter ions with defined m/z values are released from the labels, and their intensities reflect the relative abundances of the corresponding peptides in each sample.
(5) Data analysis and quantification: Relative protein expression differences among samples are quantified based on reporter ion intensity ratios.
Core Advantages of the iTRAQ Labeling Technique
1. Multiplexed Quantification Improving Analytical Throughput
A major advantage of iTRAQ lies in its ability to simultaneously quantify up to 8 or 10 biological samples within a single experiment. Compared with single-sample analytical approaches, iTRAQ markedly improves experimental throughput while reducing batch-to-batch variation.
2. Synchronous Analysis Reducing Experimental Variability
Because labeled samples are mixed and processed collectively through identical analytical workflows, the technique minimizes variation arising from differential sample handling, thereby enhancing data consistency and comparability across samples.
3. High Analytical Sensitivity Suitable for Complex Biological Matrices
Coupling iTRAQ labeling with high-resolution mass spectrometry platforms (e.g., Orbitrap or QTOF) enables detection and quantification of low-abundance proteins, membrane-associated proteins, and tissue-specific proteins. The technique is well suited to complex sample matrices such as plasma, brain tissue, and tumor tissue. Using nano-flow LC-MS/MS platforms and optimized sample pre-processing workflows, MtoZ Biolabs has achieved high proteome coverage, facilitating the identification of biologically relevant regulatory factors.
4. Compatibility with Multi-Dimensional Bioinformatics Analyses
Data generated from iTRAQ experiments exhibit favorable normalization properties and can be readily integrated with other omics datasets (e.g., metabolomics and transcriptomics) to support systems biology research and functional network analysis.
MtoZ Biolabs: Support for High-Quality iTRAQ Experimental Design and Implementation
Although iTRAQ is a robust platform for quantitative proteomics, it imposes stringent requirements on experimental precision, mass spectrometry instrumentation, and data analysis expertise. MtoZ Biolabs offers one-stop quantitative proteomics services encompassing:
Through multi-omics integration, customized research solutions are available, including iTRAQ plus metabolomics joint analysis and iTRAQ plus phosphorylation modification studies.
Despite the emergence of next-generation labeling systems such as TMT, iTRAQ remains a mature, stable, and multiplex-capable technique that retains utility in specific proteomic study contexts. MtoZ Biolabs provides technical support and flexible implementation strategies to facilitate efficient research progression and dissemination.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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