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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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