iTRAQ/TMT: Isotope Labeling and Tandem Mass Spectrometry Quantification
iTRAQ/TMT (isotope labeling and tandem mass spectrometry quantification analysis) rely on the principle of using isotope tags to label proteins, enabling the simultaneous comparison of multiple samples in mass spectrometry analysis. iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and TMT (Tandem Mass Tag) are widely used isotope labeling and quantification techniques in tandem mass spectrometry. These methods allow researchers to conduct both relative and absolute quantification of proteins in complex biological samples.
The iTRAQ technology labels the amino groups of proteins with isotope-labeled reagents, generating isotope-labeled peptides. These labeled samples are then pooled and analyzed using tandem mass spectrometry to obtain quantitative data. While iTRAQ is commonly used, TMT operates similarly, with different reagents and chemical structures that typically enable higher multiplexing and greater precision. Combining iTRAQ/TMT allows the analysis of multiple samples in a single experiment, enhancing experimental efficiency and improving data comparability.
The application of iTRAQ/TMT lies in their ability to achieve high-throughput and high-precision protein quantification, making them indispensable tools in fields such as medical research, drug development, and agricultural biotechnology. For instance, iTRAQ/TMT can help researchers examine differences in protein expression within disease tissues, facilitating the identification of potential biomarkers. These technologies are also instrumental in studying drug efficacy and toxicity, guiding new drug development. Furthermore, in agricultural biotechnology, iTRAQ/TMT are utilized to monitor protein changes in plants under various environmental conditions, providing valuable data for crop improvement.
iTRAQ Technology
1. Principle
The iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) method employs 4 or 8 isotopically labeled tags to specifically label peptide amino groups, enabling accurate quantification. iTRAQ reagents consist of a reporter group, a balance group, and a peptide-reactive group. The reporter groups vary in mass (e.g., 113Da, 114Da), while balance groups ensure equal molecular weights for different reagents. The peptide-reactive groups attach the reagents to the peptides. After labeling, peptides appear as a single peak in the primary mass spectrum due to their identical mass. In the secondary mass spectrum, the balance groups are lost, and the reporter groups produce ions with distinct m/z ratios, reflecting the relative abundance of labeled peptides.
2. Characteristics
iTRAQ can compare protein levels across up to 8 samples with labeling efficiency up to 97%. It supports automated, high-efficiency separation via SCX-HPLC and can identify up to 6000 proteins with high resolution on mass spectrometry platforms, including low-abundance and other specialized proteins. Additionally, iTRAQ is not limited by species specificity.
3. Experimental Workflow
Proteins are first enzymatically digested, typically with trypsin. The resulting peptides are labeled with distinct iTRAQ reagents and mixed. Liquid chromatography coupled with mass spectrometry is then used to perform primary and secondary mass spectrometry analysis. The resulting data undergoes bioinformatics analysis for both qualitative and quantitative assessments.
TMT Technology
1. Principle
The TMT (Tandem Mass Tag) method is similar to iTRAQ, using 2, 6, 10, or 16 isotopic tags to label peptide amino groups. TMT reagents consist of a mass reporter group, a mass-normalization component, and a reactive group. In the primary mass spectrum, labeled peptides from different samples exhibit the same m/z ratio, while in the secondary mass spectrum, reporter groups generate distinct reporter ion peaks. The intensities of these peaks reflect the relative expression of peptides in each sample, while fragment ion m/z ratios provide peptide sequence information, enabling both qualitative and relative quantification.
2. Characteristics
TMT enables higher throughput, allowing the simultaneous labeling of up to 16 different biological samples in one experiment. The smallest molecular mass difference between reporter groups is 6/1000Da, providing enhanced resolution and minimizing experimental errors.
3. Experimental Workflow
The first step involves protein extraction and quality control to ensure sample purity. TMT reagents are then used to label the peptides, which are subsequently separated by HPLC. Mass spectrometry analysis follows, and the resulting data undergoes bioinformatics processing to obtain both qualitative and quantitative results for protein expression.
Isotope labeling and tandem mass spectrometry quantification significantly enhance research efficiency by enabling the simultaneous analysis of multiple samples. With precise isotope labeling and advanced mass spectrometry, iTRAQ/TMT provide highly accurate protein quantification results. MtoZ Biolabs offers premium iTRAQ/TMT isotope labeling services, providing precise and efficient analysis of complex biological samples. We look forward to collaborating with you to drive scientific progress.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
