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.

    Related Services

    iTRAQ/TMT/MultiNotch Quantitative Proteomics Service

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