Quantitative protein profiling by mass spectrometry using isotope-coded affinity tags: Methods Mol.Biol.

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TypeArticle
Journal titleMethods Mol.Biol.
Volume439
Pages225240; # of pages: 16
Subjectanalysis; Canada; chemical; Chromatography,High Pressure Liquid; complex; COMPLEXES; database; Fractionation; IDENTIFICATION; isolation & purification; Isotopes; mass spectrometry; MASS-SPECTROMETRY; metabolism; method; Methods; Mixture; MS; protein; Proteins; Proteomics; SAMPLES; selective; Tandem Mass Spectrometry
AbstractA 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, fractionation 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
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedNo
NRC numberHAQQANI2008A
NPARC number9360650
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Record identifier84935aae-f0fc-4246-b391-90f1392a6f3e
Record created2009-07-10
Record modified2016-05-09
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