What Is the iTRAQ Method of Proteomics?
Proteomics research involves the identification and characterization of proteins to elucidate their function and interactions with other proteins. Since the composition of protein mixtures may vary across different cell types and may change under certain physiological conditions, one of the goals is often to quantify the up- or down-regulation of individual proteins. Shotgun proteomics approaches enable identification of proteins that are up-regulated or down-regulated under specific conditions and this can be studied in different cell and tissue lysates. Isobaric tags for relative and absolute quantification (iTRAQ) make it possible to both identify and quantify proteins simultaneously. iTRAQ can easily be multiplexed, enabling analysis of up to 8 different samples within the same experiment,that allows for high-throughput quantitative proteomics analysis.
What Is iTRAQ Technology
iTRAQ utilizes isobaric reagents to label the primary amines of peptides and proteins. The iTRAQ reagents usually consist of an N-methyl piperazine reporter group, a balance group, and an N-hydroxy succinimide ester group that is reactive with the primary amines of peptides. The balance groups present in each of the iTRAQ reagents function to make the labeled peptides from each sample isobaric and the quantification is facilitated through analysis of reporter groups that are generated upon fragmentation in the mass spectrometer. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides from different samples/treatments. These samples are then pooled and usually fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry (MS/MS).
Advantages of iTRAQ-based Proteomics Analysis
1. Multiplexing Capability
One of the key advantages of the iTRAQ method is its ability to multiplex, allowing for the simultaneous analysis of multiple samples in a single experiment. By using different iTRAQ reagents with distinct reporter ion masses, up to 10 or more samples can be combined and analyzed together. This significantly reduces experimental time, cost, and technical variability, making it an efficient choice for large-scale proteomic studies.
2. Relative and Absolute Quantification
iTRAQ enables both relative and absolute quantification of proteins. Relative quantification provides insights into changes in protein expression levels between different conditions or time points. Absolute quantification, on the other hand, allows for the determination of protein abundance in absolute terms, facilitating comparisons across samples.
3. Improved Accuracy and Reproducibility
The iTRAQ method offers improved accuracy and reproducibility compared to traditional label-free approaches. By incorporating isobaric tags, iTRAQ minimizes experimental variations and normalizes potential errors arising from sample preparation, digestion, and instrument performance. This enhances the reliability of quantitative proteomics data and enables more robust statistical analysis.
4. Wide Range of Applications
iTRAQ-based proteomics analysis has found applications in various biological and clinical research areas. It has been utilized in biomarker discovery, studying protein expression changes in disease progression, drug response, and therapeutic efficacy. iTRAQ has also been employed in elucidating protein interactions, investigating signaling pathways, and unraveling the dynamics of protein complexes.
What Is The Difference Between iTRAQ and TMT
|Tagging Chemistry||iTRAQ uses a four-plex or eight-plex set of isobaric tags, while TMT offers more multiplexing options ranging from two-plex to sixteen-plex.|
|Reporter Ion Intensity||In iTRAQ, the reporter ions are quantified based on their intensity ratios, while TMT uses the relative peak areas of the reporter ions for quantification.|
|Compatibility with Mass Spectrometry||iTRAQ has been used with various mass spectrometry instruments including Orbitrap and triple quadrupole systems. TMT is commonly used with Orbitrap-based mass spectrometers.|
|Cost Considerations||iTRAQ reagents are more cost-effective for lower multiplexing, while TMT reagents may be advantageous for larger-scale experiments due to their higher multiplexing capacity.|
|Compatibility with Downstream Analysis||iTRAQ and TMT have specific data analysis pipelines and software that can handle their respective quantification data. Compatibility with downstream analysis tools should be considered when choosing the technique.|
What Can We Do for You
Creative Proteomics offers protein quantification service suited for unbiased untargeted biomarker discovery. Relative quantification of proteins for biomarker discovery in complex mixtures by mass spectrometry can easily and quickly be achieved using iTRAQ technology. iTRAQ is ideally suited for comparing normal, diseased, and drug-treated samples, time course studies, biological replicates and provides relative quantitation. In addition, we also provide TMT Proteomics service, which can analyze 10 samples at a time, which is especially suitable for simultaneous comparison between multiple groups of samples and dynamic detection of biological processes.
iTRAQ workflow (4-plex) is shown above. Samples to be quantified are prepared under various treatment conditions followed by cell lysis to extract proteins. After using a standard protein assay to estimate the protein concentration of each sample, proteins are digested using an enzyme, such as trypsin, to generate proteolytic peptides. Each peptide digest is labeled with a different iTRAQ reagent and then the labeled digests are combined into one sample mixture. The combined peptide mixture is analyzed by LC-MS/MS for both identification and quantification.
A database search is then performed using the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated.
Advantage of our iTRAQ technique
- Cutting-edge facilities & optimized protocols
- High sensitivity
- Untargeted approach for biomarker discovery
- Post-translational modification is detectable
- Functional annotation and enrichment analysis
- Clustering analysis
- Network analysis
- Statistical analysis
FAQ of iTRAQ
- Why should I use iTRAQ?
In 2D LC mass spectrometry with iTRAQ all proteins are analysed, identified and quantified in one experiment, whereas in gels every spot must be imaged, its intensity measured, and then the spot cut out and identified individually.
Note: iTRAQ analysis cannot be performed on unknown genome.