iTRAQ/TMT Quantitative Proteomics Steps
Sample Preparation
1. Protein Extraction
Total proteins are extracted from various biological samples.
2. Protein Quantification
Protein concentrations are determined using methods such as BCA or Bradford assays.
Protein Digestion
Proteins are enzymatically digested using trypsin or other enzymes, cleaving at specific residues like lysine or arginine, to generate peptides.
Peptide Labeling
Peptides are chemically labeled with iTRAQ or TMT reagents following the manufacturer’s protocols. Each label corresponds to a different sample or treatment condition.
Mixing of Labeled Peptides
Labeled peptides from different samples are mixed in defined ratios to facilitate simultaneous analysis in a single experiment.
Peptide Separation
High-Performance Liquid Chromatography (HPLC) is employed to separate mixed peptide samples, reducing the complexity for subsequent MS analysis.
Mass Spectrometry Analysis
MS/MS is utilized to identify and quantify peptides using a mass spectrometer. The relative abundance of different labels is measured to compare protein expression levels across samples.
Data Analysis
Professional software such as MaxQuant or Proteome Discoverer is used for data processing and analysis. This includes identifying peptides and proteins, quantifying their relative abundance, and conducting statistical analyses and bioinformatics interpretation.
Result Validation (Optional)
Key proteins can be validated using techniques such as Western blotting or ELISA.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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