A Detailed Workflow of TMT-Based Quantitative Proteomics
TMT-based quantitative proteomics is an advanced technique employed for the quantitative analysis of protein expression. This method utilizes specific chemical tags to label protein samples, enabling comparative analysis of protein expression across different samples via mass spectrometry. The standard experimental workflow for TMT-based quantitative proteomics comprises the following steps:
1. Sample Preparation
(1) Protein Extraction: Proteins are extracted from cells, tissues, or biological fluids.
(2) Protein Quantification: Protein concentrations are measured using BCA, Bradford, or other protein quantification assays.
2. Protein Digestion
Enzymatic Digestion: Proteins are enzymatically cleaved into peptides, typically using trypsin.
3. TMT Labeling
Peptide Labeling: Peptides are labeled with TMT reagents, with each sample labeled using a distinct TMT tag.
4. Mixing of Labeled Peptides
Sample Pooling: The labeled peptides from different samples are combined to allow simultaneous analysis.
5. Liquid Chromatography Separation
Peptide Separation: The 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 by tandem mass spectrometry. The TMT tags enable relative quantification of peptides across samples by detecting reporter ions during MS/MS analysis.
7. Data Processing and Analysis
(1) Mass Spectrometry Data Processing: Specialized software such as Proteome Discoverer or MaxQuant is used to process the mass spectrometry data, identify peptides and proteins, and perform quantitative analysis.
(2) Bioinformatics Analysis: Differentially expressed proteins are further analyzed through functional annotation, pathway enrichment, and network analysis.
8. Validation Experiments
Validation Analysis: Key findings are validated using techniques such as Western blotting or ELISA.
TMT-based quantitative proteomics provides a precise and comprehensive approach for analyzing protein expression differences, particularly in complex biological samples. This technique is widely applied in studies of disease mechanisms, drug action pathways, and biomarker discovery.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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