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Quantitative Protein Profiling by Mass Spectrometry Using Isotope-Coded Affinity Tags

  • Arsalan S. Haqqani
  • John F. Kelly
  • Danica B. Stanimirovic
Part of the Methods in Molecular Biology™ book series (MIMB, volume 439)

Abstract

A key issue in proteomics is to quantify changes in protein levels in complex biological samples under different conditions. Traditional two-dimensional gel (2-DE) electrophoresis-based proteomic approaches are tedious and suffer from several limitations, including difficulties in detecting low abundant and insoluble proteins. Isotope-coded affinity tagging (ICAT®), one of the most employed chemical isotope labeling methods, can address many of the shortcomings of 2-DE. ICAT relies on the sensitivity of mass spectrometry (MS) to quantify relative protein abundance in a mixture of two differentially labeled protein samples. We describe here a detailed protocol for ICAT-based quantification of proteins in two or more biological samples, including sample preparation, ICAT labeling, frac-tionation and purification, and analysis by MS. For the MS analysis, we describe a “targeted” approach, which includes quantification of the samples using MS followed by selective identification of only the differentially expressed ICAT pairs using tandem MS (MS/MS). This approach gives more biologically relevant information than a data-dependent MS/MS analysis. We also describe the steps in data analysis, statistical analysis, and protein database searching.

Keywords

quantitative protein profiling isotope-coded affinity tag tandem mass spectrometry 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Arsalan S. Haqqani
    • 1
  • John F. Kelly
    • 1
  • Danica B. Stanimirovic
    • 1
  1. 1.Institute for Biological SciencesNational Research CouncilOttawaCanada

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