What is iTRAQ?
iTRAQ, short for Isobaric Tags for Relative and Absolute Quantitation, stands as a landmark innovation in the field of proteomics.
The journey of iTRAQ began in the early 2000s when researchers recognized the limitations of traditional proteomics approaches, such as two-dimensional gel electrophoresis and stable isotope labeling by amino acids in cell culture (SILAC). These methods, while valuable, were hampered by issues such as limited dynamic range, low throughput, and difficulty in quantifying protein abundance accurately across multiple samples.
In response to these challenges, scientists embarked on a quest to develop a novel technique that would overcome these limitations and revolutionize quantitative proteomics. The breakthrough came with the invention of iTRAQ by scientists at Applied Biosystems (now part of SCIEX) in the mid-2000s. Leveraging advances in mass spectrometry and chemical labeling technologies, iTRAQ introduced a groundbreaking approach to quantifying proteins based on isobaric tags.
The development of iTRAQ involved meticulous optimization of labeling chemistries, mass spectrometry workflows, and data analysis algorithms to ensure accurate and reproducible results. Over the years, iTRAQ has undergone continuous refinement and improvement, with new reagent designs, multiplexing strategies, and software tools enhancing its performance and versatility.
Today, iTRAQ has become the method of choice for quantitative proteomics analysis in diverse research areas, ranging from basic biology to clinical diagnostics. Its widespread adoption underscores the transformative impact of technological innovation on advancing our understanding of the proteome and its role in health and disease.
Select Services
What is the principle of iTRAQ method of proteomics?
The working principle of iTRAQ is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digest with isobaric tags. These tags are designed in such a way that they are mass-balanced, meaning they are indistinguishable during the initial mass spectrometry analysis. This allows peptides from different samples, each labeled with a different iTRAQ reagent, to be combined and simultaneously analyzed.
The iTRAQ technology uses four or eight distinct isobaric tags containing the same total mass, which allows multiplexing of samples, thereby significantly increasing throughput and minimizing experimental variability. Upon fragmentation during tandem mass spectrometry, the tags release reporter ions of different masses that provide a relative quantification of the peptides and hence the proteins from which they originated.
The working principle enables iTRAQ to simultaneously identify and quantify proteins in multiple samples, providing a comprehensive, systematic, and high-throughput method for comparative proteomic analysis. This process makes iTRAQ a powerful tool in biomarker discovery and proteomic research.
Experimental workflow of iTRAQ
The experimental workflow of iTRAQ embodies a carefully orchestrated series of steps, each meticulously designed to ensure the accurate quantification of proteins across multiple samples. This workflow represents the culmination of decades of research and development, reflecting the relentless pursuit of excellence in proteomics analysis.
Sample Preparation
At the outset, the success of an iTRAQ experiment hinges upon the meticulous preparation of biological samples. Whether derived from cell cultures, tissues, or bodily fluids, samples must undergo rigorous processing to extract proteins and prepare them for downstream analysis. This typically involves homogenization, protein extraction, and enzymatic digestion to generate a complex mixture of peptides ready for labeling.
Labeling
Central to the iTRAQ workflow is the process of labeling peptides with isobaric tags, which imparts unique mass signatures to each sample while maintaining chemical equivalence. This step, often performed using commercially available iTRAQ reagents, involves the chemical derivatization of peptide N-termini and lysine residues with isotopically encoded reporter groups. The result is a set of labeled peptides, each carrying a distinct mass tag that enables multiplexed analysis of multiple samples within a single experiment.
Fractionation
To enhance the depth and coverage of proteome analysis, labeled peptides may undergo fractionation prior to mass spectrometry analysis. Fractionation techniques such as liquid chromatography separate peptides based on their physicochemical properties, such as hydrophobicity, size, or charge. This step serves to reduce sample complexity, mitigate ion suppression effects, and improve detection sensitivity by separating peptides into distinct subsets for sequential analysis.
Mass Spectrometry Analysis
The crux of iTRAQ-based proteomics lies in the accurate quantification of labeled peptides using tandem mass spectrometry (MS/MS). In this phase, labeled peptides are subjected to fragmentation within the mass spectrometer, yielding a series of fragment ions whose relative intensities reflect the abundance of their parent peptides. By measuring the reporter ion intensities corresponding to each iTRAQ tag, researchers can quantify protein abundance across multiple samples with high precision and accuracy.
Data Interpretation
The final step in the iTRAQ workflow entails the interpretation and analysis of mass spectrometry data to extract meaningful biological insights. This process involves the identification of peptides and proteins, quantification of their abundance ratios, and statistical analysis to discern significant changes in protein expression between experimental conditions. Advanced bioinformatics tools and algorithms play a crucial role in processing raw data, performing normalization, and generating interpretable results that inform subsequent hypothesis-driven research.
Workflow of iTRAQ analysis in a rat model of CMS (Abudouwayiti et al., 2021)
What is the difference between iTRAQ and TMT?
While both iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and TMT (Tandem Mass Tags) are isobaric labeling techniques used for quantitative proteomics, they exhibit distinct characteristics and are tailored to different experimental needs.
Chemical Composition and Design
One fundamental difference between iTRAQ and TMT lies in their chemical composition and design. iTRAQ reagents typically consist of four or eight isobaric tags, each containing a reporter group, a balance group, and a mass normalization group. In contrast, TMT reagents comprise ten distinct isotopic labels, each coupled with a unique mass reporter group. This difference in design influences the multiplexing capacity and labeling efficiency of each technique, with TMT offering a higher degree of multiplexing compared to iTRAQ.
Multiplexing Capacity
Another distinguishing feature between iTRAQ and TMT is their multiplexing capacity, i.e., the number of samples that can be simultaneously analyzed within a single experiment. iTRAQ typically supports four-plex and eight-plex labeling schemes, allowing researchers to compare protein expression across four or eight different samples in parallel. In contrast, TMT offers greater multiplexing capacity with up to ten distinct channels, enabling the simultaneous analysis of ten samples within the same experiment. This higher multiplexing capacity makes TMT particularly well-suited for large-scale comparative proteomics studies involving multiple experimental conditions or sample types.
Labeling Efficiency and Specificity
The efficiency and specificity of peptide labeling represent critical considerations when comparing iTRAQ and TMT. While both techniques are capable of labeling peptides with high efficiency, differences in labeling chemistry and reagent design may impact the extent of labeling across different peptide populations. iTRAQ reagents predominantly target primary amines, including peptide N-termini and lysine side chains, for labeling, whereas TMT reagents can label both primary amines and sulfhydryl groups, thereby offering a broader scope of labeling targets. This difference in specificity may influence the quantification accuracy and coverage of each technique, particularly for peptides with specific amino acid residues.
Fragmentation Patterns and Quantification Accuracy
The fragmentation patterns generated during tandem mass spectrometry (MS/MS) analysis also differ between iTRAQ and TMT, influencing the quantification accuracy and reliability of each technique. While both methods produce characteristic reporter ions upon peptide fragmentation, variations in reagent structure and fragmentation chemistry may give rise to differences in ion intensities and spectral quality. These differences can impact the accuracy of quantification, particularly for low-abundance peptides or complex sample matrices.
Applications and Experimental Considerations
Ultimately, the choice between iTRAQ and TMT depends on the specific experimental requirements, sample complexity, and research objectives. iTRAQ is well-suited for comparative proteomics studies involving fewer samples and requiring high-throughput analysis, whereas TMT offers greater multiplexing capacity and broader labeling specificity, making it ideal for large-scale experiments and in-depth characterization of complex proteomes. Additionally, factors such as cost, instrument compatibility, and data analysis considerations may also influence the selection of one technique over the other.
Select Services
What is the difference between ICAT and iTRAQ?
Labeling Strategy
One fundamental difference between ICAT and iTRAQ lies in their labeling strategies and chemistries. ICAT utilizes a differential isotopic labeling approach, wherein cysteine residues within peptides are selectively labeled with light or heavy isotopic tags. This strategy enables the quantification of cysteine-containing peptides with high specificity, making ICAT particularly well-suited for targeted analysis of specific subsets of proteins or post-translational modifications.
In contrast, iTRAQ employs an isobaric labeling strategy, wherein peptides from different samples are labeled with distinct isotopic tags containing reporter and balance groups. This approach allows for multiplexed quantification of peptides across multiple samples within a single experiment, thereby enabling comparative analysis of protein expression levels across diverse experimental conditions or sample types.
Multiplexing Capacity
Another key difference between ICAT and iTRAQ is their multiplexing capacity. ICAT typically supports only two-channel labeling, with peptides from control and experimental samples labeled with light and heavy isotopic tags, respectively. In contrast, iTRAQ offers higher multiplexing capacity, with four-plex and eight-plex labeling options available. This greater multiplexing capability allows researchers to analyze multiple samples simultaneously, thereby increasing experimental throughput and efficiency.
Targeted vs. Global Analysis
The choice between ICAT and iTRAQ often depends on the nature of the proteomics experiment and the research objectives. ICAT is well-suited for targeted analysis of specific proteins or pathways, as it enables selective labeling of cysteine-containing peptides and facilitates quantification of changes in protein abundance or modification status. In contrast, iTRAQ is more suitable for global proteomics analysis, as it allows for comprehensive quantification of protein expression levels across the entire proteome without bias towards specific amino acid residues.
Sensitivity and Coverage
Both ICAT and iTRAQ offer advantages in terms of sensitivity and proteome coverage. ICAT's selective labeling of cysteine residues ensures high specificity and sensitivity for detecting changes in targeted proteins or modifications. However, its coverage may be limited by the presence of non-cysteine-containing peptides or proteins in the sample. In contrast, iTRAQ's multiplexing capability enables simultaneous analysis of a larger number of peptides and proteins, thereby enhancing proteome coverage and enabling the detection of low-abundance proteins or subtle changes in protein expression levels.
Select Services
Reference
- Abudouwayiti, Aihaidan, et al. "iTRAQ-based quantitative proteomic analysis of the improved effects of total flavones of Dracocephalum Moldavica L. in chronic mountain sickness." Scientific Reports 11.1 (2021): 17526.
