ITRAQ TMT (Isotope Labeling Quantification)

Isotope labeling quantification:

iTRAQ (isobaric Tags for Relative and Absolute Quantitation) &TMT (Tandem Mass Tags) The peptide in vitro labeling and quantification technology developed by AB Sciex and Thermo respectively uses multiple isotopic reagents to label the N-terminus or lysine side chain groups of protein peptides. Different molecular weight reagents are used to label samples from different sources, thereby achieving a comparison of protein profiles between different samples.

ITRAQ experimental steps:

1. Sample preparation: Quantitatively calculate the protein concentration of the sample to be tested;

2. Protein reduction alkylation and enzymatic hydrolysis: Take 100 μ g of each sample from each group and process it according to the iTRAQ kit requirements. After enzymatic hydrolysis with sequencing grade trypsin solution, collect the hydrolyzed peptide segments by ultrafiltration tube centrifugation;

3. iTRAQ labeling: Equilibrate the ITRAQ reagent to room temperature, dissolve it in 150 μ L isopropanol, take the enzymatically hydrolyzed peptide and add it to the iTRAQ reagent. After 2 hours of reaction at room temperature, add water to terminate the reaction. Mix equal amounts of different groups of samples and vacuum freeze dry the samples

4. LC-MS-MS analysis: Perform tandem mass spectrometry identification using Thermo's Q-Exactive Orbitrap.

5. Data processing and bioinformatics analysis

Result display:

completebureauAnalyze clustering heatmap

*Statistical analysis of protein count for 0

Experimental cycle and delivery standards

1. Experimental period: 4-6 weeks

2. Delivery standards: protein quantification results, identification results, mass spectrometry raw data, bioinformatics analysis results (G0 analysis) KEGG analysis, etc

Information to be confirmed

1. Species of the sample

2. Type of sample (tissue or cell or blood)

3. Sample quantity and grouping

4. Analysis requirements

References

[1]XuX，Zhu Q，Zhang R，et al. ITRAQ-Based Proteomics Analysis of Acute Lung Injury Induced by Oleic Acid in

Mice[J]. Cellular Physiology and Biochemistry, 2017:1949-1964.

[2]QuZ, GaoF , LiL, et al. Label-Free Quatitative Proteomic Analysis of Diferentially Expressed Membrane Proteins

of Pulmonary Alveolar Macrophages Infected with Highly Pathogenic Porcine Reproductive and Respiratory Syndrome

Virus and Its Attenuated Strain[J]. PROTEOMICS, 2017:1700101.

ELISA kit free testing and experimental outsourcing quotation