Protocol abstract
Nature Protocols 1, - 139 - 145 (2006)
Published online: 27 June 2006 | doi:10.1038/nprot.2006.22
Subject Categories: Biochemistry and protein analysis | Genomics and proteomics | Isolation, purification and separation | Spectroscopy and structural analysis
Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry
Yuzuru Shiio1 & Ruedi Aebersold2,3
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.
- Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.
- The Institute for Molecular Systems Biology, ETH-Zurich, Eidgenössische Technische Hochschule-Zurich and Faculty of Sciences, University of Zurich, Zurich, Switzerland.
- The Institute for Systems Biology, Seattle, Washington 98103, USA.
Correspondence to: Ruedi Aebersold2,3 e-mail: rudolf.aebersold@imsb.biol.ethz.ch
nature-products
A-Z product listing
- Cleavable ICAT reagents(Applied Biosystems)
- Cleaving reagent A(Applied Biosystems)
- Cleaving reagent B(Applied Biosystems)
- column(Polymicro Technologies)
- Dithiothreitol(Sigma-Aldrich)
- HPLC columns(polyLC Inc)
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