Isotope-Coded Affinity Tagging ICAT

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 

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

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Von Haller, P.D., Yi, E., Donohoe, S., Vaughn, K., Keller, A., Nesvizhskii, A.I., Eng, J., Li, X.J., Goodlett, D.R., Aebersold, R., Watts, J.D. (2003). The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-coded Affinity Tag (ICAT) and Tandem Mass Spectrometry II. Evaluation of Tandem Mass Spectrometry Methodologies for Large-Scale Protein Analysis, and the Application of Statistical Tools for Data Analysis and Interpretation. Mol. Cell. Proteomics 2, 428 -42. 

Fauq, A.H., Kache, R., Khan, M.A., and Vega, I.E. (2006) Synthesis of acid-cleavable light isotope-coded affinity tags (ICAT-L) for potential use in proteomic expression profiling analysis. Bioconjugate Cbem. 17, 248-254. 

D.K. Han, J. Eng, H. Zhou and R. Aebersold, Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags (ICAT) and mass spectrometry, Nat. Biotech., 19 (2001) 946-951. 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology... 

More importantly insoluble proteins, such as membrane-bound and nuclear proteins, are under-represented within 2DE analysis. However, membrane proteins can be identified by MS following a IDE separation. Unfortunately, hydrophobicity is the most common reason for poor representation of abundant proteins and seems set to be with us for quite some time (Santoni et al., 2000 Rabilloud, 2002). Emerging proteomic methods without the use of 2DE are being developed, such as isotope-coded affinity tagging (ICAT) and multi-dimensional protein identification technology (MuDPIT). 

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... 

Yan W, Lee H, Deutsch EW, Lazaro CA, Tang W, Chen E, et al. A dataset of human liver proteins identified by protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry. Mol Cell Proteomics 2004 3(10) 1039-1041. 

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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