Cleavable ICAT® Reagents

Increase Proteome Coverage Now you can increase proteome coverage, identify and quantify the proteins that matter and accelerate your discovery efforts. The specific, cleavable label reduces sample complexity enabling quantitation and identification of medium to low abundant proteins from complex samples. The incorporation of the mass difference, a result of tagging, enables true expression-dependent experiments where quantitation is achieved in MS mode and only those proteins that exhibit change are further analyzed via MS/MS for identification.

Improved PerformanceCleavable ICAT reagents incorporate a number of key technological advances that significantly improve protein-expression analysis. Incorporation of an acid-cleavable linker into the ICAT molecule allows removal of the biotin affinity tag before MS and MS/MS analysis. This improves MS/MS performance and significantly increases the number of proteins identified and quantified with higher confidence scores in a single experiment. Incorporation of ¹³C into the heavy ICAT reagent molecule promotes co-elution of the heavy and light isotopes in reversed phase chromatography, thereby increasing accuracy of quantification measurements by LC-ESI-MS, LC-MALDI-TOF-MS, and MALDI-TOF-MS. These significant improvements in the reagent design, along with the availability of ICAT software for data analysis, enable researchers to fully exploit the potential of this technology in protein-expression profiling studies.
 

ICAT Reagent CompositionThe cleavable ICAT reagent is composed of four main segments: 1) A protein reactive group (Iodocetamide): The reactive group covalently links the isotope-coded affinity tag to the protein by alkylation of free cysteines. 2) An affinity tag (biotin): This simplifies the analysis of the ICAT reagent-labeled peptides by selecting and concentrating the cysteine-containing peptides, thereby reducing the complexity of the peptide mixture. 3) An isotopically labeled linker (C₁₀H₁₇N₃O₃): The linker chain can substitute up to nine ¹³C atoms. Chemically the light and heavy molecules are identical-they merely differ in mass. The heavy reagent with nine ¹³C atoms is nine Daltons heavier than the light reagent. Mass spectrometric comparison of peptides labeled with heavy and light reagents provides a ratio of the concentration of the proteins in the original sample.4) An acid cleavage site: Following avidin affinity purification of the ICAT reagent-labeled peptides, you can remove the biotin portion of the label and part of the linker by adding TFA. This reduces the overall mass of the tag on the peptides and improves the overall peptide fragmentation efficiency.
The cleavable ICAT® reagents are available in either Methods Development Kit or the 10-Assay Kit formats or as bulk reagents.

For Research Use Only. Not for use in diagnostic procedures.