Isotope-coded affinity tag labeling

Mass spectrometry — It has excellent sensitivity but low throughput and is semiquantitative. The ICAT (isotope-coded affinity tag) labeling method for proteins enables differential display analyses using mass spectrometry (Griffin and Abersold, 2001). This may be... 

Smolka MB, Zhou H, Purkayastha S, Aebersold R. 2001. Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis. Anal Biochem 297 25-31. 

Smolka M, Zhou H, Aebersold R. Quantitative protein profiling using two-dimensional gel electrophoresis, isotope-coded affinity tag labeling, and mass spectrometry. Mol Cell Proteomics 2002 1 19-29. 

The other approach to quantitative protein profiling is based on stable isotope labelling of proteins and peptides and automated tandem mass spectrometiy (MS/MS) (Gygi et al., 1999b). This method, termed isotope-coded affinity tag labelling or ICAT, has been shown to be robust, reproducible and amenable to high throughput automation (Ideker et al.,... 

In vitro labeling of proteins using isotope-coded affinity tags... 

The isotope-coded affinity tag approach utilizes chemical labeling that allows quantitation when combined with mass spectrometry. ICAT is desirable because the chemical labeling takes advantage of the mass defects of monoisotopic stable isotopes. ICAT uses an ICAT reagent to differentially label protein samples on their cysteine residues. ICAT is advantageous because it permits the evaluation of low-abundance proteins and proteins at both extremes of molecular weights and isoelectric points.60... 

For proteins of low abundance as well as membrane proteins, 2D PAGE suffers from a lack of resolving power. Another possibility, besides chromatography, to circumvent the limitations of 2D PAGE is to use chemically labeled tags, such as two different isotopes (ICAT, isotope-coded affinity tag) (Adam, 2002). A suitable... 

The use of isotope-coded affinity tag (ICAT) approaches for identification and quantitation of proteins contained in complex mixtures is a developing methodology that bypasses 2-DGE (Gygi et al., 1999). The ICAT approach is a derivative of an isotope dilution method and involves the introduction of a postgrowth biotin affinity tag onto cysteine residues via iodoacatamide chemistry (Figure 6.4). Proteome samples obtained from two cellular states are labeled one with the d0-ICAT and the other with d8-ICAT. The two... 

Labeling proteins with heavy and light tags and screening the hit compound versus an inactive control, followed by mass spectrometric comparison of the two samples, is another approach that avoids many of the common pitfalls in affinity methods (3). Techniques such as stable isotope labeling with amino acids in cell culture and isotope-coded affinity tagging (ICAT) exemplify these techniques. 

The isotope-coded affinity tag (ICAT) technique involves differential labeling of two different protein populations on the side chain of reduced cysteinyl residues using one of two chemically identical but isotopically different ICAT reagents (19). By incorporating a biotin affinity tag into the ICAT reagents, selective isolation and purification of labeled peptides substantially reduces sample complexity. The ICAT approach has been applied successfully to the systematic identification and quantification of proteins contained in the microsomal fraction of cells... 

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