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Type: Artigo de periódico
Title: Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis
Author: Smolka, MB
Zhou, HL
Purkayastha, S
Aebersold, R
Abstract: The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate. (C) 2001 Academic Press.
Country: EUA
Editor: Academic Press Inc
Citation: Analytical Biochemistry. Academic Press Inc, v. 297, n. 1, n. 25, n. 31, 2001.
Rights: fechado
Identifier DOI: 10.1006/abio.2001.5318
Date Issue: 2001
Appears in Collections:Unicamp - Artigos e Outros Documentos

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