Detailed Process of TMT Quantitative Proteomics Experiment
TMT (Tandem Mass Tag) quantitative proteomics is an advanced technology used for quantitatively analyzing protein expression. This method labels protein samples with specific chemical tags and then uses mass spectrometry to compare protein expression differences between different samples. The basic workflow of a TMT quantitative proteomics experiment is as follows:
1. Sample Preparation
- Protein Extraction: Proteins are extracted from cells, tissues, or biological fluids.
- Protein Concentration Measurement: The protein concentration is determined using methods such as BCA, Bradford, or other protein quantification assays.
2. Protein Digestion
- Enzymatic Digestion: Trypsin is commonly used to digest proteins into peptides.
3. TMT Labeling
- Peptide Labeling: Peptides are labeled with TMT reagents. Different samples are labeled with different TMT tags.
4. Mixing Labeled Peptides
- Sample Mixing: Labeled peptides from different samples are mixed together for simultaneous analysis.
5. Liquid Chromatography Separation
- Peptide Separation: Mixed peptides are separated using High-Performance Liquid Chromatography (HPLC) or Ultra-Performance Liquid Chromatography (UPLC).
6. Mass Spectrometry Analysis
- MS/MS Analysis: Peptides are analyzed using a mass spectrometer. TMT tags allow for quantitative comparison of peptides in different samples by detecting reporter ions in MS/MS analysis.
7. Data Processing and Analysis
- Mass Spectrometry Data Processing: Specialized software (such as Proteome Discoverer, MaxQuant, etc.) is used to process mass spectrometry data, identify peptides and proteins, and perform quantitative analysis.
- Bioinformatics Analysis: Analyze protein expression differences, annotate biological functions, and perform pathway and network analysis, etc.
8. Validation Experiments
- Validation Analysis: Key findings are validated using techniques such as Western blot, ELISA, etc.
Figure 1. Workflow of TMT-based Proteomics Service
TMT quantitative proteomics provides a precise and comprehensive method for analyzing protein expression differences, especially in complex biological samples. This technology finds broad applications in disease mechanism research, drug action mechanism analysis, and biomarker discovery.
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