How Can iTRAQ Labeling Technology Be Applied in Peptidomics Analysis?
- Cells/tissues: washed with cold PBS to minimize protein degradation
- Body fluids: supplemented with protease inhibitors and centrifuged rapidly to remove cell debris
- Endogenous peptidomics: molecular weight cutoff (<10 kDa), solid-phase extraction, or C18 bead enrichment
- Controlled enzymatic digestion: applied to generate short peptides for studying protein regulation and functional fragments
- Reporter group: releases identifiable ions in MS/MS (e.g., m/z 114, 115, 116)
- Balance group: maintains equal overall mass
- Reactive group: covalently binds to peptide N-termini and lysine residues
- Equal loading: each sample normalized to the same total peptide amount (>100 µg recommended)
- Reconstitution: peptides dissolved in iTRAQ buffer, avoiding ammonium-containing solutions such as Tris
- Labeling: incubation with iTRAQ reagents at 37℃ for 1–2 h
- Quenching: addition of a stop reagent to terminate residual activity
- Mixing: labeled samples combined in equal proportions before subsequent steps
- C18 SPE columns remove salts and labeling by-products
- Optional magnetic bead enrichment concentrates peptides in the target size range
- Data-dependent acquisition (DDA) is commonly used for peptide identification and quantification.
- Alternatively, data-independent acquisition (DIA) and parallel reaction monitoring (PRM) can be applied to enhance reproducibility and enable targeted validation.
- Differential peptides are mapped to their corresponding proteins, followed by pathway annotation using Gene Ontology (GO), KEGG, and Reactome.
- This analysis supports subsequent biomarker validation, predictive modeling, and mechanistic interpretation.
In peptidomics analysis, mass spectrometry (MS) is commonly employed to identify endogenous peptides naturally present in biological samples. However, information on peptide presence alone is insufficient to elucidate disease mechanisms or to discover reliable biomarkers. Quantitative analysis, particularly the comparative assessment of differential expression across multiple samples, is essential for understanding pathological processes and identifying potential therapeutic targets. iTRAQ Labeling Technology, which is widely applied in proteomics, has also emerged as an important tool in specialized peptidomics research.
What is iTRAQ Labeling Technology? Principles and Advantages
iTRAQ is a multiplexed relative quantification method based on isobaric isotope tagging. By attaching chemically identical tags to peptides from different samples, labels that have the same overall mass but yield distinct reporter ions upon fragmentation, multiple samples can be simultaneously compared and quantified within a single MS analysis.
Core Features of iTRAQ Technology
(1) Multiplexing capacity: supports 4-plex and 8-plex, enabling parallel analysis of up to eight groups of samples.
(2) Isobaric labeling: the tags do not affect chromatographic separation or MS1 mass-to-charge detection.
(3) MS/MS-based quantification: reporter ions released during tandem MS provide quantitative information.
The iTRAQ 8-plex platform is frequently employed for comparing peptide expression profiles across groups such as disease subtypes, pre- and post-treatment states, or different physiological conditions, offering an efficient tool for high-throughput screening.
Experimental Workflow: Standard Application of iTRAQ Labeling Technology in Peptidomics
1. Sample Collection and Peptide Extraction
Appropriate extraction strategies are chosen according to the study aim and sample type (tissue, serum, urine, cerebrospinal fluid, etc.):
(1) Source
(2) Extraction Method
All steps are conducted under low-temperature, protective conditions to preserve peptide structures and post-translational modifications.
2. iTRAQ Labeling
(1) Principle
Each iTRAQ tag consists of three components:
(2) Workflow
If labeling is performed before enrichment, peptide N-termini must remain unblocked to ensure labeling efficiency.
3. Cleanup, Fractionation, and Processing
(1) Cleanup
(2) Fractionation
High-pH reversed-phase liquid chromatography (RPLC) separates mixed samples into 8–12 fractions, reducing complexity and increasing peptide identification.
4. LC-MS/MS Analysis
(1) Analytical Platform
A nanoflow liquid chromatography (nanoLC) system coupled with a high-resolution mass spectrometer (e.g., Orbitrap Exploris 480) is employed.
Tandem mass spectrometry (MS/MS) using higher-energy collisional dissociation (HCD) enables the accurate release of iTRAQ reporter ions.
(2) Acquisition strategy
5. Data Processing and Quantitative Analysis
MtoZ Biolabs utilizes specialized software and in-house databases to perform the following key analyses:
(1) Peptide Identification
MS/MS spectra are matched using software such as Proteome Discoverer, MaxQuant, and PEAKS, with compatibility for major protein databases including UniProt, RefSeq, and Ensembl.
(2) Reporter Ion Intensity Extraction
For each peptide, iTRAQ reporter ion peak intensities are extracted to calculate relative abundance ratios, with corrections applied for background noise and reporter ion interference (cross-talk).
(3) Statistical Analysis
Differential peptides are identified based on fold change, p-value, and false discovery rate (FDR).
Visualization of expression profiles is achieved using heatmaps, principal component analysis (PCA), clustering, and volcano plots.
(4) Functional Enrichment and Pathway Annotation
Applicability of iTRAQ Labeling Technology in Peptidomics: A Conditionally Affirmative Answer
Although iTRAQ is primarily applied to enzymatically digested peptides in proteomics, it can also be adapted for the quantification of endogenous peptides in specific peptidomics studies. Its applications mainly include:
1. Differential Expression Analysis of Small Endogenous Peptides
Examples include neuropeptides, hormones, and immune-regulatory peptides. These naturally occurring short peptides can be directly labeled to assess expression changes across experimental conditions.
2. Quantitative Studies of Enriched Peptide Fractions
Peptide mixtures obtained through pretreatment methods such as molecular weight cutoff or magnetic bead enrichment are particularly suitable for iTRAQ labeling and quantification, a strategy frequently employed in liquid biopsy studies.
3. Analysis of Peptide Responses in Drug Intervention and Disease Models
For instance, monitoring drug-induced changes in peptide abundance enables evaluation of their potential roles as drug targets or downstream effectors.
Advantages and Limitations of iTRAQ Labeling Technology in Peptidomics
1. Advantages
(1) High quantitative accuracy: all samples analyzed in a single MS run, minimizing batch effects.
(2) High throughput: supports multiple groups per experiment, reducing cost and time.
(3) Data integration: facilitates combined analysis with proteomic datasets to build protein–peptide interaction maps.
(4) Versatility: suitable for endogenous peptide analysis in serum, urine, and cerebrospinal fluid.
2. Limitations
(1) Reduced applicability for very short peptides lacking reactive sites (N-terminal or lysine).
(2) Quantification accuracy depends on high labeling efficiency; incomplete labeling introduces errors.
(3) Unsuitable for full HLA peptidomics, as labeling may disrupt structural integrity and binding studies.
In conclusion, iTRAQ labeling technology remains a valuable tool for parallel quantification of multiple groups and for investigating endogenous peptide expression. With careful experimental design and optimized sample preparation, iTRAQ can serve as a robust quantitative engine in peptidomics. Continued development of mass spectrometry platforms and labeling strategies will further support biomarker discovery and mechanistic exploration. MtoZ Biolabs will continue to enhance mass spectrometry platforms and labeling strategies to deliver reliable, efficient, and precise solutions for peptidomics quantification, thereby advancing biomarker discovery and mechanistic insights.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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