Isotope-coded protein label chemical labeling

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 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... 

One of the main problems to overcome in order to realise quantitative proteomics is that mass spectrometry is not strictly a quantitative technique with respect to molecular abundance. One approach to overcome this problem is to perform comparative experiments in parallel where one sample is labelled with an isotope (such as 0 or H) and the other is unlabelled. Mass differences between labelled and unlabelled samples may be observed in a mass spectrometer and the relative ratios quantified (Figure 9.21). Obviously this method of quantification relies on equivalent mass expression by both samples in spite of the isotopic labelling of the one and not the other. In other words, in spite of the labelling, both samples should behave identically chemically. The simplest way to effect labelling is to use isotope-coded affinity tags (ICATs). According to this technique, two identical populations of proteins... 

Isotope-coded affinity tag (ICAT) peptide labeling is an approach that combines accurate quantification and concurrent sequence identification of individual proteins in complex mixtures (Gygi, et al 1999). This method is based on a newly synthesized class of chemical reagents used in combination with tandem mass spectrometry. The method consists of four steps ... 

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