Protein quantification analysis-iTRAQ
Isobaric tags for relative and absolute quantification, also known as i-TRAQ, is a protein quantification method using isotopes to "label" proteins from different samples, which results in different molecular weight with the same proteins in different samples.
With this method, you can analyze protein quantification between different samples at the same time
With this method, you can analyze protein quantification between different samples at the same time
After the proteins are digested with trypsin, the peptides are labeled with iTRAQ reactive group (amine specific peptide reactive group) through covalent bond on the N-end. The peptides from different are labeled with four different iTRAQ isotopes (in the picture below, four samples are labeled with iTRAQ 114-117 respectively.
The labeled peptides are mixed and analyzed through LC-MS/MS. The mass of the same peptide in four different samples are still the same due to the balance of different equilibrant groups corresponding to four different isotopes, so the mass-to-charge ratio (m/z) is still the same on the first mass spectrometry. After breakage of peptides into fragment ions, we can compare with the database to identify the protein of the peptide. We can also calculate the relative abundance of the same peptide from different samples through different isotopes (number "4" in the picture down below)
Service Specification
| Basic Analysis | Standard Analysis | Advance Analysis | |
| Specification | 1D, 120 min | 2D, 60 min * 24 fractions | 2D, 60 min * 72 fractions |
| Protein number can be quantified | 100-1000 | 500-3000 | 500-6000 |
Note 1: We can compare 2-10 samples.
Note 2: For phosphoproteome, the protein number can be quantified is dependent on the level of the phosphorylation in your sample.
Quantitative Proteome Quantitative 
phosphoproteome
phosphoproteome 
Working day not including delivery time: 3-5 weeks
Quantitative proteome: Basic/Standard/Advanced analysis
(a) Sample type: Cell pellet, Protein extracts (please provide buffer constituents)
(b) Sample amount: ≥5 ug (Basic), ≥10 ug (Standard), ≥20 ug (Advanced) for each group
(c) Sample concentration: ≥0.5 ug/uL (Basic), ≥1 ug/uL (Standard), ≥2 ug/uL (Advanced)
(d) Limitations: Detergents (≤0.2%), Urea (≤1 M); Buffer without Tris-Base, Ammonium Salt, Gly-gly, DTT, Mercaptoethanol
(e) Platform: online-2DLC LTQ-Orbitrap MS
Quantitative phosphoproteome: Basic/Standard/Advanced analysis
(a) Sample type: Cell pellet, Protein extracts (please provide buffer constituents)
(f) Sample amount: ≥10 ug (Basic), 40ug (Standard), 100 ug (Advanced) for each group
(b) Sample concentration: ≥0.5 ug/uL (Basic), ≥1 ug/uL (Standard), ≥2 ug/uL (Advanced)
(c) Limitations: Detergents (≤0.2%), Urea (≤1 M); Buffer without Tris-Base, Ammonium Salt, Gly-gly, DTT, Mercaptoethanol
(d) Platform: online-2DLC LTQ-Orbitrap MS
(a) Sample type: Cell pellet, Protein extracts (please provide buffer constituents)
(b) Sample amount: ≥5 ug (Basic), ≥10 ug (Standard), ≥20 ug (Advanced) for each group
(c) Sample concentration: ≥0.5 ug/uL (Basic), ≥1 ug/uL (Standard), ≥2 ug/uL (Advanced)
(d) Limitations: Detergents (≤0.2%), Urea (≤1 M); Buffer without Tris-Base, Ammonium Salt, Gly-gly, DTT, Mercaptoethanol
(e) Platform: online-2DLC LTQ-Orbitrap MS
Quantitative phosphoproteome: Basic/Standard/Advanced analysis
(a) Sample type: Cell pellet, Protein extracts (please provide buffer constituents)
(f) Sample amount: ≥10 ug (Basic), 40ug (Standard), 100 ug (Advanced) for each group
(b) Sample concentration: ≥0.5 ug/uL (Basic), ≥1 ug/uL (Standard), ≥2 ug/uL (Advanced)
(c) Limitations: Detergents (≤0.2%), Urea (≤1 M); Buffer without Tris-Base, Ammonium Salt, Gly-gly, DTT, Mercaptoethanol
(d) Platform: online-2DLC LTQ-Orbitrap MS
Please click the link below for the specification of Proteomics service
Sample requirement
Quantitative proteome
1. Summary report. pdf
2. Protein-peptide report (Identification result and Raw ratio). xlsx
3. Normalized Protein Ratio (Global normalization). xlsx
4. Differential Protein (List of differentially expressed proteins). xlsx
5. Pathway Enrichment Analysis (For Standard and Advanced analysis). xlsx
Quantitative phosphoproteome
1. Summary report. pdf
2. Protein-Phosphopeptide Report (Identification result and Raw ratio). xlsx
3. Normalized Phosphopeptide Ratio (Global normalization). xlsx
4. Differential Phosphopeptide (List of differentially expressed proteins). xlsx
5. Pathway Enrichment Analysis (For Standard and Advanced analysis). xlsx
1. Summary report. pdf
2. Protein-peptide report (Identification result and Raw ratio). xlsx
3. Normalized Protein Ratio (Global normalization). xlsx
4. Differential Protein (List of differentially expressed proteins). xlsx
5. Pathway Enrichment Analysis (For Standard and Advanced analysis). xlsx
Quantitative phosphoproteome
1. Summary report. pdf
2. Protein-Phosphopeptide Report (Identification result and Raw ratio). xlsx
3. Normalized Phosphopeptide Ratio (Global normalization). xlsx
4. Differential Phosphopeptide (List of differentially expressed proteins). xlsx
5. Pathway Enrichment Analysis (For Standard and Advanced analysis). xlsx
Q1. How many samples can iTRAQ identify at the same time?
A: The maximum sample iTRAQ can identify is four. For samples more than four, we have Tandem Mass Tag (TMT), which can identify as much as ten samples at the same time.
Q2. Can you analyze protein quantification between phosphorylated proteins?
A: Yes, we can analyze protein quantification between phosphorylated proteins (Phosphoproteomic)
Q3. What will be the limitation of serum or plasma samples?
A: (a) Abundant protein removal must be done to prevent the results of the identification to be mostly
abundant proteins in serum or plasma sample
(b) The number of proteins in the blood is far less than other tissues, so the proteins that can be identified will be limited, at most 1000 proteins.
Q4. What does peptide confidence mean in the raw data?
A: Basically it is related to the accuracy of precursor m/z, the similarity of fragment ion and predicted fragment ion and the calculation of false discovery rate (for more detailed information please refer to the basic settings of Mascot)
Q5. Each sample has a variability in the raw data (e.g 115/117 variability)，what is the relationship between this value and 115/117 count?
A: Count means how many spectra have the 115/117 ratio and the variability means the variation of the 115/117 ratio among these counts. If the count number is more than 10, you could ignore the variability. If the count number is less than 5 and the variability is higher than 100%, that means the ratio may not be reliable.
Q6. What does PEP (Posterior error probabilities of the peptide) mean in the raw data?
A: PEP means the probability that the observed PSM is incorrect.
Q7. What does confidence (CID and HCD) mean?
A: CID and HCD are methods to break the peptides, both are used in identifying peptides. Confidence means the confidential rate of using these two methods to identify the peptides respectively (According to the q-value of PEP and FDR).
Q8. How many proteins can be identified?
A: For proteome
1. Basic analysis: 100-1000
2. Standard analysis: 500-3000
3. Advanced analysis: 500-6000
(Phosphoproteomic is different according to the phosphorylated condition of the proteins)
Q9. Can you do this service with blood samples?
A: If you do protein identification, we suggest you combine with abundant protein removal service to exclude the high abundant proteins (e.g. albumin). If you don't the results will be affected greatly. For protein quantification, we suggest label-free quantification. For further details, please contact our technician.
Q10. Is cytokine-related proteins be identified using LC-MS/MS system?
A: If you wish to detect the cytokine in cell supernatant, we do not suggest you use LC-MS/MS platform. For further details please contact our technician.
A: The maximum sample iTRAQ can identify is four. For samples more than four, we have Tandem Mass Tag (TMT), which can identify as much as ten samples at the same time.
Q2. Can you analyze protein quantification between phosphorylated proteins?
A: Yes, we can analyze protein quantification between phosphorylated proteins (Phosphoproteomic)
Q3. What will be the limitation of serum or plasma samples?
A: (a) Abundant protein removal must be done to prevent the results of the identification to be mostly
abundant proteins in serum or plasma sample
(b) The number of proteins in the blood is far less than other tissues, so the proteins that can be identified will be limited, at most 1000 proteins.
Q4. What does peptide confidence mean in the raw data?
A: Basically it is related to the accuracy of precursor m/z, the similarity of fragment ion and predicted fragment ion and the calculation of false discovery rate (for more detailed information please refer to the basic settings of Mascot)
Q5. Each sample has a variability in the raw data (e.g 115/117 variability)，what is the relationship between this value and 115/117 count?
A: Count means how many spectra have the 115/117 ratio and the variability means the variation of the 115/117 ratio among these counts. If the count number is more than 10, you could ignore the variability. If the count number is less than 5 and the variability is higher than 100%, that means the ratio may not be reliable.
Q6. What does PEP (Posterior error probabilities of the peptide) mean in the raw data?
A: PEP means the probability that the observed PSM is incorrect.
Q7. What does confidence (CID and HCD) mean?
A: CID and HCD are methods to break the peptides, both are used in identifying peptides. Confidence means the confidential rate of using these two methods to identify the peptides respectively (According to the q-value of PEP and FDR).
Q8. How many proteins can be identified?
A: For proteome
1. Basic analysis: 100-1000
2. Standard analysis: 500-3000
3. Advanced analysis: 500-6000
(Phosphoproteomic is different according to the phosphorylated condition of the proteins)
Q9. Can you do this service with blood samples?
A: If you do protein identification, we suggest you combine with abundant protein removal service to exclude the high abundant proteins (e.g. albumin). If you don't the results will be affected greatly. For protein quantification, we suggest label-free quantification. For further details, please contact our technician.
Q10. Is cytokine-related proteins be identified using LC-MS/MS system?
A: If you wish to detect the cytokine in cell supernatant, we do not suggest you use LC-MS/MS platform. For further details please contact our technician.