Increase proteome coverage: By labeling protein cysteinyl groups with the cleavable ICAT® Reagent, you can increase proteome coverage, identify and quantify proteins of interest, and accelerate your discovery efforts. The specific, cleavable label enables quantitation and identification of medium- to low-abundance proteins and reduces sample complexity. Incorporating the mass difference through tagging enables true, expression-dependent experiments, where quantitation is achieved in MS mode, and only those proteins that exhibit mass changes are further analyzed via MS/MS for identification.
Improved performance: Cleavable ICAT® Reagents incorporate a number of key technological advances that significantly improve protein-expression analysis. Incorporation of an acid-cleavable linker into the ICAT® Reagent 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, so that higher confidence scores can be provided in a single experiment. Incorporation of carbon-13 into the heavy ICAT® Reagent molecule promotes co-elution of protein pairs labeled with heavy and light isotope-coded tags 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 composition: The cleavable ICAT® Reagent is composed of four moities: 1) a protein reactive group (iodocetamide) that covalently links the isotope-coded affinity tag to the protein through cysteine alkylation; 2) a biotin affinity tag that simplifies labeled-peptide analysis through affinity chromatography, specifically selecting for cysteine-containing peptides; 3) either a C13-labeled linker chain (heavy) or non-labeled chain (light, 9 dalton mass difference) that allows for mass spectrometric comparison of heavy- and light-labled peptides and provides a ratio of the concentration of the proteins in the original sample; and 4) an acid cleavage site allows for the removal of the biotin label following affinity purification of the labeled peptides to reduce the overall tag mass for improvement in peptide fragmentation efficiency.
The cleavable ICAT® Reagents are available through the Methods Development Kit, 10-Assay Kit formats, or as bulk reagents.