iTRAQ Mass Spectrometry
iTRAQ mass spectrometry is an advanced analytical technique primarily utilized in proteomics to achieve both relative and absolute quantification of proteins. By employing isotopic tags that chemically react with amino acids in peptides, this method enables the precise measurement of protein levels across multiple samples within a single mass spectrometry run. The key principle of iTRAQ mass spectrometry lies in isotopic labeling, which generates identical mass signals for the same proteins across different samples. During collision-induced dissociation (CID), these tags release reporter ions with distinct masses, facilitating quantitative comparisons. This powerful approach has broad applications in biomedical research, drug development, and the study of complex biological processes.
In biomedical research, iTRAQ mass spectrometry is widely applied to identify proteins associated with disease mechanisms and progression. By analyzing proteomic differences between healthy and diseased samples, researchers can uncover potential biomarkers for diagnosis and therapeutic intervention. For instance, comparing the serum proteomes of cancer patients with those of healthy individuals can reveal differentially expressed proteins, offering new insights into diagnostic and prognostic tools. In drug discovery, iTRAQ mass spectrometry is instrumental in studying drug action mechanisms. It enables researchers to quantitatively analyze global protein expression profiles in cells or tissues before and after drug treatment, identifying potential drug targets and elucidating mechanisms of action. This accelerates the development of new therapeutics. Furthermore, iTRAQ mass spectrometry is used in disease mechanism research, particularly in understanding neurodegenerative diseases and autoimmune disorders. This technique provides critical insights into disease pathology and aids in identifying novel therapeutic targets, forming the foundation for personalized medicine.
Workflow of iTRAQ Mass Spectrometry
1. Sample Preparation
Proteins are extracted from biological samples while ensuring the integrity and purity of the samples. Extracted proteins are enzymatically digested into peptides, preparing them for subsequent isotopic labeling and analysis.
2. Isotopic Labeling
Peptides from different samples are chemically labeled using iTRAQ reagents. These isotopic tags provide unique mass signatures for each sample, allowing simultaneous comparative analysis. The labeled peptides are then combined into a single mixture for mass spectrometry analysis.
3. Liquid Chromatography Separation
The mixed peptide samples are separated using high-performance liquid chromatography (HPLC) to improve resolution. This step ensures that peptides are effectively separated, enhancing the accuracy of subsequent mass spectrometry analysis.
4. Mass Spectrometry Analysis
The separated peptide fractions are analyzed using a mass spectrometer. During CID, the isotopic tags release reporter ions with unique masses, enabling the quantification of peptides in different samples. Advanced software is used to process and interpret the mass spectrometry data, providing protein identification and quantification results.
Limitations of iTRAQ Mass Spectrometry
1. Labeling Efficiency and Sample Loss
The chemical labeling process in iTRAQ mass spectrometry can be influenced by various factors, potentially leading to incomplete labeling or sample loss. These issues may compromise the accuracy of quantification and increase experimental complexity.
2. Signal Interference and Data Complexity
The isotopic tags used in iTRAQ labeling may introduce signal interference during mass spectrometry, especially when analyzing highly complex samples. This increases the difficulty of data interpretation, requiring additional computational resources and time for analysis.
3. High Cost
The cost of iTRAQ reagent kits and specialized instrumentation can be a limitation for budget-constrained laboratories. Furthermore, the intricate sample preparation and data analysis processes contribute to additional labor and time expenses.
MtoZ Biolabs specializes in providing professional iTRAQ mass spectrometry services, along with other tag-based quantification techniques such as TMT and SILAC. Our team of experts offers comprehensive support, ranging from experimental design to data analysis, ensuring reliable and precise results. By collaborating with us, researchers can focus on their scientific objectives without being burdened by technical complexities. We are dedicated to delivering high-quality services that support advancements in proteomics research.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?