1 / 21

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. Outline. Protein Quantification ICAT-based Approach Implementation Conclusion. Protein Quantification (I).

octavius-soto
Download Presentation

Kuen-Pin Wu Institute of Information Science Academia Sinica

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. Outline • Protein Quantification • ICAT-based Approach • Implementation • Conclusion 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 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 Kuen-Pin Wu, IIS, Academia Sinica

  5. ICAT-based Approaches Kuen-Pin Wu, IIS, Academia Sinica

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

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

  10. Implementation(Chromatogram Reconstruction) Kuen-Pin Wu, IIS, Academia Sinica

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

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

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

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

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

  20. 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 Kuen-Pin Wu, IIS, Academia Sinica

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

More Related