Two or more different samples are labeled at the
intact protein level with isotope-coded tags.
1.2 The samples are combined for digestion, and the labeled peptides are specifically separated by affinity chromatography against one part of the label (biotin-avidin enrichment).
1.3 These peptides are then analyzed by LC-MS/MS together.
1.4 The ratios of signal intensities of differentially mass-tagged peptide pairs are quantified to determine the relative levels of proteins from these two samples.
Figure 1 Procedure of Relatively Quantitative Proteomics with ICAT
2.1 ICAT has the advantage of labeling intact proteins prior to digestion, which can reduce system variability comparing to some proteomics techs.
Figure 2 Comparisons among Quantitative Proteomics Methods
2.2 ICAT was developed to reduce the sample complexity and identify low-abundance proteins and peptides in complex samples.
Because of the sulfhydryl-reactive chemical group, only free thiols on cysteine residues are labeled with this tag. The sample is then passed over immobilized avidin, which binds to the biotin tag and purifies the labeled peptides from the sample.
Because not all peptides have cysteine residues, this method does not result in global labeling and thus is an inherent approach to reduce sample complexity.
3. Shortcoming Comparing to iTRAQ Reagent and TMT Reagent
3.1 Does not result in global labeling. ICAT labeling does have a bias against proteins and peptides that lack cysteine residues, which is considerable compared to proteins that lack lysine residues. For example, 14% of Escherichia coli (E. coli) open reading frames (ORFs) do not code for cysteines, while only 0.8% do not code for lysine (although half of those could still be tagged because of terminal amines). This difference in amino acid availability should be considered when determining the right isotopic labeling method to use for quantitative proteomics analyses.
3.2 Although affinity purification of ICAT-labeled peptides reduces sample complexity by 10-fold, the cysteine-specific labeling method also reduces protein sequence coverage by the same factor.
Because of this limitation, isotope-coded protein labeling (ICPL) was developed, in which lysine residues and available N-termini on intact proteins are isotopically labeled with a heavy (d4) or light (d0) tag. This approach increases the level of labeling, because significantly more terminal amino groups are available than cysteine resides. Also, ICPL is amenable to a greater level of pre-MS fractionation than other labeling methods, because sample complexity can be reduced at both the protein level (before digestion; electrophoresis or LC) and the peptide level (after digestion; LC). ICPL also allows the simultaneous comparison of three experimental conditions in a single experiment with two heavy tags (d7 and d3) and the d0 light tag.
This multiplex capability distinguishes ICPL from ICAT and the other labeling methods listed above.
4. ICAT tags
ICAT technology is a gel-free method for quantitative proteomics that relies on chemical labeling reagents.
4.1 These chemical probes consist of three general elements:
4.1.1 A reactive group capable of labeling a defined amino acid side chain (e.g. iodoacetamide to modify cysteine residues)
4.1.2 An isotopically coded linker
4.1.3 A tag (e.g. biotin) for the affinity isolation of labeled proteins/peptides
For the quantitative comparison of two proteomes, one sample is labeled with the isotopically light (d0) probe and the other with the isotopically heavy (d8) version.
Figure 3 Structures of the ICAT Tags
4.2 Development of ICAT tags:
4.2.1 A newer version of acid labile labels has been developed, which has improved the chromatographic performance of the process.
4.2.2 The original tags were developed using deuterium, but later the same group redesigned the tags using 13C instead to circumvent issues of peak separation during LC. As known to all, the deuterium can interact with the stationary phase of the column.