A quantitative tool for comparative protein expression

Isotope Coded Affinity Tagging is a strategy for using MS and MS/MS data to concurrently identify and quantify comparative protein expression within complex mixtures.

The method employs chemical reagents termed isotope-coded affinity tags (ICATs) to create cysteine thiol group derivatives of proteins, the digest peptides of which exhibit a mass difference equal to that of the hydrogen and deuterium substituted ICAT reagents. The initial ICAT reagent has hydrogen and deuterium variants which confer an 8 Da mass difference on their respective thiol derivatives.

The isotopically variant digest peptide derivatives show little or no difference in chromatographic behaviour while the isotopic mass distinction is clearly evident in the MS and MS/MS spectra, enabling identification and quantification of the differentially tagged parent protein in a single MS run.

Protein mixtures from two different sources are respectively treated with hydrogen and deuterium ICAT variants which covalently bond to every cysteinyl residue. After the protein mixtures are combined and proteolysed to peptides, the ICAT-labelled peptides are isolated utilising the biotin component of the ICAT molecule. The peptides are then separated by microcapillary HPLC.

Each pair of ICAT-labelled peptides essentially coelutes because they are virtually chemically identical. MS/MS spectrometry of fragment ions identifies the ICAT-reagent linked peptide's amino acid sequence while the relative abundance of parallel peaks with the 8 Da separation provides quantification of the expression of the parent proteins from the different sources.

By treating expressed proteins with the two isotopically variant ICATs, it is possible to accurately assay and compare protein expression from different sample sources and under a range of experimental conditions.

Proteins can be reacted with the ICAT reagent in the presence of stabilising agents such as urea and sodium dodecyl sulphate (SDS), thereby keeping them in solution until they are enzymatically digested.

The presence of a biotin moiety in the ICAT reagent and subsequently tagged protein derivative enables selective enrichment of cysteine-containing peptides by avidin affinity column chromatography, thereby significantly reducing the complexity of the peptide mixture generated by concurrent digestion of multiple proteins. Avidin affinity purification of the tagged peptides has the additional benefit of eliminating contaminants incompatible with mass spectrometry.

Alternative techniques for quantitative protein expression analysis, such as 2D gel separation followed by scintillation counting, visual imaging or mass spectrometry, is limited to the measurement of highly abundant proteins due to restrictions in the sample size which can be loaded on the gel. ICAT analysis has no such limitation.

In addition, the technique is compatible with all fractionation methods that can reduce mixture complexity and enrich for proteins of low abundance. The accuracy of ICAT quantification is demonstrated by the ability to obtain accurate measurements of small changes in relative protein levels.

Using an ion trap MS, the ICAT method is able to quantify up to a 100-fold difference in expression levels for low abundance digest peptides and determine even larger differences for more abundant peptides.