Post-harvest chemical stable isotope labelling

11th December 2014

Posted By Paul Boughton

ICAT reagents structure and reactivity
ICAT reagents workflow

Osama Chahrour and John Malone look at post-harvest chemical stable isotope labelling methods in mass spectrometry-based quantitative proteomics

Stable isotopes quantitative proteomics identify equivalent peptides or peptide fragments utilising the specific increase in mass due to mass tags with stable isotopes. The common workflow is to tag protein or peptide samples with equivalent reagents, one of which includes a heavy mass tag and the other a light mass or no tag. The labelled samples are analysed by mass spectrometry after being mixed and fractionated. The peaks in the mass spectra reveal the ratio of the two different isotopic or mass tag variants. The ratio is then used to identify protein or peptide relative abundances. Several variants of the technique can be implemented, which are presented below1, 2.

Isotope-coded affinity tags (ICATs) are biotinylated iodoacetamide derivatives (or acrylamide derivatives) that react with the sulfhydryl groups of denatured peptide side chains. The intermediate bridge of these reagents, contains the isotopic carbon or hydrogen atoms used for differential labelling, connecting iodoacetamide to a biotin group for affinity capture of the derivatised peptides onto a streptavidin based column.

The biotin-streptavidin affinity capture allows cysteine-containing peptides to be isolated from the complex sample mixture, thereby considerably reducing the number of different peptides/molecules introduced into the mass spectrometer ion source. The linker bridge is available in two forms, one normal/light version and one heavy form, in which hydrogen or carbon atoms are replaced by deuterium/13C.

Two protein mixtures representing two different cell states (sick cell vs healthy cell) are treated with the light (healthy) or heavy (sick) ICAT tags (Fig. 2). The labelled protein mixtures are then quantitatively combined and proteolysed. Peptides binding an ICAT tag are selectively isolated by streptavidin columns and analysed by mass spectrometer. The relative abundance of the healthy/sick states of the peptide is determined by the ratio of signal intensities of the heavy/light tagged peptide pairs3. The major disadvantage of ICAT reagents is that, approximately 10% of proteins do not have a cysteine residue and therefore are excluded from this type of analysis1,4.

The ICPL technology incorporates isotope labelling of free amino groups of intact proteins. ICPL reagents label lysine side chains based on the unique ability of N-hydroxysuccinimide (NHS)-nicotinic acid ester derivatives to fully derivatise primary amino groups. Therefore, the method is suitable for different kinds of protein samples, including tissues extracts or body fluids. Similar in principle to ICAT labelling, ICPL reagents also have variants with different numbers of deuterium atoms to allow multiplex quantitative analysis. For example, the ICPL Quadruplex method used ICPL_0, ICPL_4 (4 Deuteriums), ICPL_6 (6 13C) and ICPL_10 (4 Deuteriums + 6 13C) reagents allowing the simultaneous quantitation of four independent samples1.

Non-isobaric tagging, ie the three labelling methods discussed previously produce versions of each analysed peptide with mass variation at a particular amount. The two forms will appear as two peaks with a certain mass difference in the mass spectrum. The mass-difference model is limited to a binary (2-plex), ternary (3-plex) or tertiary (4-plex) set of tags. Because of the limited plex numbers, comparison of multiple states cannot be achieved in one experiment. Thus, a multiplexed set of reagents for quantitative protein analysis has been developed.

Isobaric tagging is achieved by using chemical moieties or tags which are identical in mass to labelled peptides so that all derivatised peptides are isobaric, chromatographically indistinguishable and yielding a single peak in the mass spectrum for both samples (sick and non-sick samples). However, the relative abundances of the isobarically tagged peptides are revealed when the moieties fragment during MS/MS experiment to release reporter ions with different masses5.

The amine-reactive group of the tag (N-Hydroxysuccinimide ester-activated compounds) covalently binds to the peptide amino terminus and free amino termini of lysine residues of peptides and proteins with high efficiency. In addition to the reactive group, the isobaric mass tags are designed to include two parts: a reporter or signature region and a mass normalising region. The total mass of the reporter region and the normalising moieties is the same in all versions of the isobaric reagent, but the individual weights change.

Consequently, when the reporter ion is released in MS/MS spectrum it can be correlated to a particular sample source6.

Two isobaric tag families are commercially available. The first is the Tandem Mass Tag (TMT) reagent family which includes TMTzero, TMTduplex (reporting group mass 126 and 127 Da), TMTsixplex (reporting group mass 126 to 131 Da) and TMT10plex sets that are designed for a rapid and cost-effective evolution from method development to high-throughput protein quantitation. 

The mass reporter part is segregated from a mass normalisation moiety via a fragmentation vulnerable linker. The TMTzero tag is suitable for testing and optimisation of sample preparation, fractionation and mass spectrometric fragmentation for peptide identification through reporter detection without using the more costly isotope-labelled tags1.

The other family is isobaric tags for absolute and relative quantification (iTRAQ), with up to 8-plex versions available with the following reporter ions/isobaric tag values: 113/192, 114/191, 115/190, 116/189, 117/188, 118/187, 119/186, and 121/184 ensuring that all tags have a same total mass.


Quantitative mass spectrometry protein analysis allows the comparison of protein expression between different states of biological systems. The recent advances in this field combined with bioinformatics technologies, which are required to interpret complex data, are vital for biomarker discovery and drug target validation and will lead to clinical advantages for years to come.

Osama Chahrour and John Malone are with Almac Sciences, Craigavon, Northern Ireland, UK. 


1  Nakamura, T., and Oda, Y. (2007) Mass spectrometry-based quantitative proteomics. Biotechnol. Genet. Eng. Rev. 24, 147–164; 

2  Ong, S.-E., and Mann, M. (2005) Mass spectrometry-based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–62; 

3  Petriz, B. A., Gomes, C. P., Rocha, L. A. O., Rezende, T. M. B., and Franco, O. L. (2012) Proteomics applied to exercise physiology: a cutting-edge technology. J. Cell. Physiol. 227, 885–98; 

4  Nesvizhskii, A. I., Vitek, O., and Aebersold, R. (2007) Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat. Methods 4, 787–97.; 

5  Domon, B., and Aebersold, R. (2006) Mass spectrometry and protein analysis. Science 312, 212–7; 6. Gingras, A.-C., Gstaiger, M., Raught, B., and Aebersold, R. (2007) Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell Biol. 8, 645–54.




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