Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.
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.
Smolka
M B
MB
Departamento de Bioquímica, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil.
Zhou
H
H
Purkayastha
S
S
Aebersold
R
R
D000345:Affinity Labels / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D003545:Cysteine / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling / Q000379:methods*; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D000345:Affinity Labels; D010446:Peptide Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D007768:Lactalbumin; D014357:Trypsin; D003545:Cysteine