Proteomics is the large-scale study of proteins, which are the functional molecules within all living things. This field investigates the entire set of proteins in a cell, tissue, or organism to understand how they work together. A technique in this field is iTRAQ, which stands for Isobaric Tags for Relative and Absolute Quantitation. It is a chemical labeling method used to measure and compare the amounts of different proteins between samples to pinpoint changes in protein levels, which can provide a window into the cellular mechanics of health and disease.
How iTRAQ Labels Work
The iTRAQ method relies on small chemical tags attached to proteins once they are broken into smaller pieces called peptides. Each tag has three main parts. The first is the peptide-reactive group, which fastens the tag to the peptides in a sample.
A second component is the reporter group, which allows for measurement. In an experiment, each sample is labeled with a tag that has a unique reporter group. The final component is the balancer group, which offsets the mass of the reporter group, making the total mass of every tag identical.
This design means that when peptides from different samples are mixed, they are initially indistinguishable by mass. This concept is called isobaric labeling, where different tags have the same total weight but different internal parts. Only when the tags are fragmented during analysis are the unique reporter groups released and measured, revealing the relative quantity of each peptide.
The iTRAQ Experimental Journey
An iTRAQ experiment begins with sample collection, such as obtaining tissue from a healthy subject and a diseased one. Scientists first extract all the proteins from these samples and then use an enzyme to digest the protein chains into shorter fragments called peptides. This step is necessary because the analysis equipment is better suited to handling these smaller pieces.
Once the peptides are prepared, the labeling process begins. Peptides from each sample are mixed with a different iTRAQ reagent; for example, peptides from the healthy sample might receive the “114” tag, while the diseased sample receives the “115” tag. Following this, all labeled samples are combined into a single mixture, which minimizes experimental variations by ensuring all samples are processed together.
The combined peptide mixture is introduced into a liquid chromatography system, which separates the complex mixture into simpler fractions. These fractions are then directed into a mass spectrometer for a two-stage analysis. In the second stage, specific peptides are selected and fragmented, which breaks the iTRAQ tag and releases the unique reporter ions. The intensity of these released ions is measured, corresponding to the amount of that peptide in each of the original samples.
Unlocking Biological Insights with iTRAQ
The data from iTRAQ provides insights into complex biological systems and disease. One application is in biomarker discovery, where researchers look for proteins that show differences in abundance between healthy individuals and those with a particular condition. An iTRAQ study might identify proteins that are elevated in the blood serum of early-stage cancer patients, pointing toward potential diagnostic markers.
This technique also helps unravel disease mechanisms. By comparing protein profiles, scientists can see which cellular pathways are disrupted. In neurodegenerative diseases like Parkinson’s or Alzheimer’s, iTRAQ can reveal changes in brain proteins related to energy metabolism or cellular structure, offering clues about how the disease progresses and the molecular events driving it.
iTRAQ is also a tool in pharmacology and drug development. Researchers can treat cells with a new drug and use iTRAQ to measure the resulting changes in thousands of proteins. This can help confirm that a drug is hitting its intended target and reveal any unintended effects on other protein pathways, which helps in understanding a drug’s mechanism and identifying potential side effects.
Key Capabilities of iTRAQ
iTRAQ’s multiplexing capability is the ability to analyze multiple samples in a single experiment. Researchers can combine four to eight different samples, such as different disease stages or treatment conditions, into one analysis. This approach reduces variability compared to analyzing samples separately, leading to more reliable data and saving time.
The method provides accurate relative quantification, determining how much a protein’s level has changed between samples. For instance, it can show that a protein is twofold more abundant in a treated sample compared to an untreated control. This comparative information helps researchers understand biological responses.
While used for relative measurements, iTRAQ can be adapted for absolute quantification. This involves using standardized reference peptides to determine the precise amount of a protein in a sample. This flexibility and its applicability to a wide range of sample types make iTRAQ a widely used technique.