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Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry

Abstract

A main objective of proteomics research is to systematically identify and quantify proteins in a given proteome (cells, subcellular fractions, protein complexes, tissues or body fluids). Protein labeling with isotope-coded affinity tags (ICAT) followed by tandem mass spectrometry allows sequence identification and accurate quantification of proteins in complex mixtures, and has been applied to the analysis of global protein expression changes, protein changes in subcellular fractions, components of protein complexes, protein secretion and body fluids. This protocol describes protein-sample labeling with ICAT reagents, chromatographic fractionation of the ICAT-labeled tryptic peptides, and protein identification and quantification using tandem mass spectrometry. The method is suitable for both large-scale analysis of complex samples including whole proteomes and small-scale analysis of subproteomes, and allows quantitative analysis of proteins, including those that are difficult to analyze by gel-based proteomics technology.

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Figure 1: The ICAT strategy for quantifying differential protein expression.
Figure 2: Improving the proteome coverage by the ICAT technology.
Figure 3: Analysis of immunopurified protein complexes.
Figure 4: Typical Coomassie blue staining pattern of ICAT processing aliquots.

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Correspondence to Ruedi Aebersold.

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Shiio, Y., Aebersold, R. Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry. Nat Protoc 1, 139–145 (2006). https://doi.org/10.1038/nprot.2006.22

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