Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/194857
Type: Artigo de periódico
Title: Optimization Of The Isotope-coded Affinity Tag-labeling Procedure For Quantitative Proteome Analysis.
Author: Smolka, M B
Zhou, H
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.
Subject: Affinity Labels
Cysteine
Electrophoresis, Gel, Two-dimensional
Electrophoresis, Polyacrylamide Gel
Isotope Labeling
Lactalbumin
Mass Spectrometry
Molecular Weight
Peptide Fragments
Proteome
Saccharomyces Cerevisiae
Saccharomyces Cerevisiae Proteins
Sodium Dodecyl Sulfate
Time Factors
Trypsin
Urea
Rights: fechado
Identifier DOI: 10.1006/abio.2001.5318
Address: http://www.ncbi.nlm.nih.gov/pubmed/11567524
Date Issue: 2001
Appears in Collections:Artigos e Materiais de Revistas Científicas - Unicamp

Files in This Item:
File SizeFormat 
pmed_11567524.pdf359.27 kBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.