The field of proteomics focuses on the large-scale study of proteins, which carry out nearly all biological functions. Accurately measuring protein levels in complex biological mixtures, such as tissues or fluids, presents a significant challenge. Traditional methods often faced limitations in throughput, dynamic range, and precision. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) emerged as a powerful technique to overcome these issues. iTRAQ allows for the simultaneous quantification of proteins across multiple biological samples, offering a comprehensive view of protein changes.
The Core Mechanism of iTRAQ
iTRAQ uses specialized chemical labels called isobaric tags. Each tag consists of a reporter group, a balance group, and a peptide-reactive group. Different samples are labeled with tags that have the same overall mass, but their internal composition of isotopes varies. For instance, a 4-plex iTRAQ reagent has a total mass of 145 Da, while an 8-plex reagent has a total mass of 305 Da.
When labeled peptides from different samples are combined and analyzed by a mass spectrometer, they appear as a single peak in the initial mass spectrometry (MS) scan because their total mass is identical. This isobaric property enables the multiplexing of samples, allowing several samples to be run simultaneously. During the subsequent tandem mass spectrometry (MS/MS) step, these labeled peptides are fragmented.
Fragmentation causes the iTRAQ tags to break apart, cleaving the bond between the reporter and balance groups. This releases distinct low-mass reporter ions from each tag. The mass of each reporter ion is unique to its original sample; for example, 4-plex iTRAQ uses reporter ions from 114 to 117 Da, and 8-plex uses ions from 113 to 119 Da and 121 Da. The intensity of these reporter ions directly corresponds to the relative abundance of the peptide, and thus the protein, from each original sample.
The balance group, also released during fragmentation, ensures the overall mass of the peptide-tag combination remains consistent across all labeled samples prior to fragmentation. This allows for accurate relative quantification by comparing the intensities of the reporter ions. This enables precise measurement of protein abundance changes between different biological conditions.
Step-by-Step iTRAQ Workflow
An iTRAQ experiment begins with sample preparation, extracting proteins from biological sources like cells, tissues, or fluids. Proteins then undergo enzymatic digestion, typically with trypsin, breaking them into smaller peptide fragments suitable for labeling and mass spectrometry.
Each peptide sample, often representing different experimental conditions, is individually labeled with a unique iTRAQ reagent. These reagents covalently attach to the N-terminus and lysine residues of the peptides. After labeling, the tagged peptide mixtures from all samples are combined into a single pooled sample.
The pooled sample then undergoes separation, commonly using liquid chromatography (LC), to resolve the complex mixture of peptides. This reduces sample complexity and improves mass spectrometry data quality. The separated peptides are then introduced into a tandem mass spectrometer (MS/MS) for analysis.
In the mass spectrometer, a precursor ion scan (MS1) identifies peptides based on their mass-to-charge ratio. Selected precursor ions are then fragmented, releasing reporter ions unique to each iTRAQ tag. The intensities of these reporter ions are measured, providing the relative quantification of the peptides.
Finally, specialized software processes the mass spectrometry data. It interprets reporter ion intensities to determine the relative abundance of proteins across the original samples. The software also identifies specific proteins based on their peptide fragmentation patterns.
Key Applications in Research
iTRAQ technology has widespread utility across various research disciplines, providing insights into complex biological systems. A prominent application is in biomarker discovery for diseases, identifying proteins whose levels change significantly in diseased versus healthy states. For instance, iTRAQ helps pinpoint potential diagnostic or prognostic markers in cancers and neurological disorders by comparing protein expression profiles.
The technology also helps understand drug mechanisms. By analyzing protein changes in cells or tissues before and after drug treatment, scientists can identify protein pathways influenced by the therapeutic agent. This aids in elucidating drug targets, off-target effects, and mechanisms of drug resistance, supporting pharmaceutical development.
Beyond disease and drug studies, iTRAQ investigates how organisms respond to environmental stimuli. This includes examining protein expression changes when cells are exposed to toxins, stress, or different nutrients. Such studies contribute to understanding cellular adaptation, environmental toxicology, and nutritional science.
iTRAQ also facilitates the study of protein expression across different cell types, developmental stages, or physiological conditions. Researchers can compare proteomes of various cell lines, track protein changes during cell differentiation, or analyze how protein levels shift during normal growth and aging. This provides a detailed molecular picture of cellular identity and biological processes.
Advantages and Distinguishing Features of iTRAQ
iTRAQ offers several advantages in quantitative proteomics. Its multiplexing capability allows researchers to simultaneously analyze between four to eight different protein samples in a single experiment. This reduces experimental variability and increases throughput compared to individual sample analysis.
The technology provides high accuracy and precision in protein quantification. Labeling peptides from different samples with isobaric tags and pooling them minimizes experimental variations from separate analyses. This allows for more reliable comparisons of protein abundance across different conditions.
iTRAQ also facilitates comprehensive protein identification, enabling the quantification of thousands of proteins in a single experiment. This broad coverage provides a complete picture of protein expression changes within a biological system. The ability to obtain both relative and, in some cases, absolute protein quantification adds to its utility for detailed and high-throughput proteomic investigations.