Sixplex TMT 10-Level Proteomics
TMT (Tandem Mass Tags) technology is a widely utilized method for quantitative proteomics, enabling the simultaneous comparison of protein expression levels across multiple samples.
In a 10-plex TMT experiment, up to ten different samples can be quantitatively compared. Each TMT tag corresponds to a distinct mass, which can be accurately identified using a mass spectrometer. These tags are covalently attached to proteins or peptides, allowing samples from varying conditions or time points to be analyzed together in a single mass spectrometry run. By assessing the relative intensity of these tags, researchers can quantitatively compare the protein expression levels from different samples. The primary steps involved are as follows:
1. Sample Preparation
Proteins are extracted from biological samples obtained under different conditions or time points.
2. Protein Digestion
Proteins are enzymatically digested, typically using trypsin, to generate smaller peptides.
3. Peptide Labeling
Peptides derived from different samples are chemically labeled with distinct TMT tags.
4. Peptide Mixing and Separation
The labeled peptides are combined and then separated by high-performance liquid chromatography (HPLC) to reduce sample complexity.
5. LC-MS/MS Analysis
The peptides are analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), which facilitates the identification of peptides and their corresponding proteins. Protein expression levels are quantitatively assessed by measuring the intensity of the TMT tags.
6. Mass Spectrometry Analysis
The separated peptides were analyzed using a mass spectrometer to identify the peptides and their corresponding proteins, and the protein expression levels were quantitatively analyzed by measuring the intensity of different TMT tags.
7. Data Processing and Analysis
Specialized software tools are used for data processing, protein identification, and comparative analysis of protein abundance across samples to elucidate differential protein expression.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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