Isotope Coded-Affinity Tags (ICAT)

What is Isotope Coded-Affinity Tags (ICAT)

ICAT is a new technique for studying expressed proteome. This technique uses a new chemical reagent called isotope affinity labeling reagent to pre-selectively label a certain class of proteins. Then the labeled protein was separated and purified, and then identified by mass spectrometry (MS), and the relative abundance of its parent protein in the original cell was quantitatively analyzed according to the ratio of ionic strength of different peptide segments on the mass spectrometry.

The ICAT reagent consists of three parts:

1. The reagent reacts with the protein group, which specifically binds the sulfhydryl group of the cysteine residue in the peptide chain;
2. The middle linker can stably bind isotopes;
3. affinity tag - biotin, can bind to ovalbumin, select the isolation of ICAT-labeled peptides.

Figure 1. Structure of the ICAT reagents

The Specific Operation Process ICAT Technology

1. The two sources are closely related and different states of cell division, protein is reduced;
2. The two kinds of samples (two different states of cell division) were labeled with different ICAT reagents;
3. The two kinds of samples (two different states of cell division) after complete labeling were mixed, and the peptides of different sizes were hydrolyzed by trypsin;
4. Solid cation column exchange to remove all residual trypsin, scale remover, reducing agent and ICAT reagent;
5. Labeled and unlabeled peptides were separated by ovalbumin affinity chromatography.
6. After elution, the labeled peptide was separated again by liquid chromatography and analyzed by tandem mass spectrometry. By comparing the ionic strength of a pair of peptide peaks with the same peak type, the relative content of the unified protein in the two samples could be inferred. Because cysteine is a relatively rare amino acid, cysteine-labeled peptides provide a limited range for peptide identification when database searches are performed (Cysteines in peptides were labeled by ICAT), and therefore the corresponding proteins are easier to identify.

Figure 2. The ICAT strategy for quantifying differential protein expression

Advantages of ICAT Technology

1. Membrane proteins can be identified and quantified by protein separation at the peptide level;
2. The complexity of protein mixtures was reduced by selecting labeled cysteine-containing peptides;
3. By comparing two or more closely related protein samples from different sources, the proportion of protein expression changes in different states can be obtained. The protein content of closely related sources can be each other's internal reference standard;
4. For proteins containing multiple cysteine residues, positive identification and quantification can be obtained by repeated retrieval of peptides containing cysteine;
5. Because ICAT technology is based on chromatographic separation, any reagent that promotes proteolysis can be used;
6. Can directly identify and measure low-abundance proteins.

Application of ICAT Technology

1. Determination and quantification of membrane proteins: membrane proteins have low solubility and are easy to precipitate during conventional two-dimensional electrophoresis separation, which cannot be well separated, resulting in slow progress in membrane protein research. Scientists have used ICAT technology to systematically determine the relative content of two closely related cellular proteins, which has solved the problem of membrane protein solubility, enabled the identification and quantification of membrane proteins, and promoted the development of membrane protein research.
2. Identification and quantification of low-abundance proteins: An important measure of the resolution of any mass spectrometry method for protein identification and quantification is its ability to analyze low-abundance proteins. Two-dimensional electrophoresis combined with mass spectrometry has sufficient sensitivity but insufficient accuracy for protein analysis. ICAT technology can ensure both sensitivity and accuracy.
3. Application of ICAT technology in proteomics: Phosphorylation of protein isotope affinity tag reagents can identify the phosphoric acid sites in proteins. ICAT is also used in solid-phase isotope labeling reagent technology.

Conclusion

Although ICAT technology plays an important role in the study of expressed proteomes and is a significant progress in research methods, which can be widely used in proteome research with different targets, ICAT technology still has many defects and needs to be further improved and improved.