1. Kuen-Pin Wu Institute of Information Science Academia Sinica
Quantitative Analysis of Isotope-coded Affinity Tag (ICAT) Labeled Proteins Based on Mass Spectrometry
Kuen-Pin Wu
Institute of Information Science
Academia Sinica
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

2. Kuen-Pin Wu, IIS, Academia Sinica
Outline
Protein Quantification
ICAT-based Approach
Implementation
Conclusion
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

3. Protein Quantification (I)
Monitoring how protein expression changes.
Protein-protein interaction
Protein functions
Often, it is not necessary to measure the absolute amount of protein present but rather to compare the relative abundance of proteins in two related states.
Normal cell vs. cancer cell
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

4. Protein Quantification (II)
Usually accomplished by combining
Protein separation
Sequence identification
2DE-MS/MS
2D gel + tandem mass spectrometry (MS/MS)
HPLC-MS/MS
High performance liquid chromatography (HPLC) + MS/MS
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

5. ICAT-based Approaches
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

6. Kuen-Pin Wu, IIS, Academia Sinica
ICAT Reagent
Biotin
Linker (heavy or light)
Reactive group
Heavy reagent (d8): linker contains 8 2H
Light reagent (d0): linker contains 8 1H
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

7. ICAT-based Protein Quantification
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

8. Protein Identification
Identify proteins based on MS/MS scans
Mass spectra define peptides; peptides define proteins
Database search
Sequest
Mascot
De Novo sequencing
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

9. Chromatogram Reconstruction
Why chromatogram reconstruction?
The elution of proteins/peptides in chromatography is continuous, but only some of them will be analyzed by mass spectrometry.
Relative Abundance
Relative Abundance
Time
Time
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

10. Implementation (Chromatogram Reconstruction)
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

11. Single Ion Chromatogram Reconstruction (I)
A MS spectrum
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

12. Single Ion Chromatogram Reconstruction (II)
Smoothing by 3-point moving average
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

13. Single Ion Chromatogram Reconstruction (III)
Peak selection and background subtraction
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

14. Single Ion Chromatogram Reconstruction (IV)
Reconstructed ion chromatogram at one time point
Extracted ion chromatogram (XIC)
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

15. Single Ion Chromatogram Reconstruction (V)
XIC smoothing (B-spline), peak selection and background subtraction
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

16. Determine the Ratio of a Single Peptide Ion
Peak pair recognition
coefficient of correlation
Peak alignment
cICAT: 12C and 13C
Peak area calculation
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

17. Determine the Ratio of a Peptide
Each peptide may have several charge states (ions)
Calculate the weight of each ion ratio
A weight is a function of peak area and shape similarity
Use Dixon’s test to delete outlier(s)
The peptide ratio is expressed as a weighted sum of the ion ratios
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

18. Determine the Ratio of a Protein
Each protein may have several peptide
Select peptides that uniquely belongs to the protein
Calculate the weight of each selected peptide ratio
A weight is a function of protein identification score
Use Dixon’s test to delete outlier(s)
The protein ratio is expressed as a weighted sum of the selected peptide ratios
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

19. System Errors Elimination
We assume that most proteins have the same expression level in two cell states
Adjust final results to make the ratios of most proteins in the two cell states 1
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

20. Kuen-Pin Wu, IIS, Academia Sinica
Conclusion
Current state of ICAT-based strategy
pros and cons
Sensitive to proteins with low abundance
We have developed a fully automatic tools to perform ICAT-based protein quantification
The cICAT reagent has been patented
Other isotopes are used in different approaches
Wet-lab experiments are difficult to perform well
Other quantification approaches such as ITRAQ are developing
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica

21. Kuen-Pin Wu (巫坤品) kpw@iis.sinica.edu.tw
Thank you!
Kuen-Pin Wu (巫坤品)
2019/7/6
Kuen-Pin Wu, IIS, Academia Sinica