sample superimposes with its differentially labelled identical counterpart in the other
sample. Scanning of the gel at two different wavelengths that excite the two dye
molecules reveals whether any individual spot is associated with only one dye
molecule rather than two. Most spots will, of course, fluoresce at both wavelengths,
but if a spot is associated with only one dye molecule then this tells us that that
protein can have been present in only one of the extracts, and the wavelength at
which it fluoresces tells you which extract it was originally in.8.5.2 Isotope-coded affinity tags (ICAT)
Isotope-coded affinity tags (ICAT) uses mass spectrometry (rather than 2-D gels) to
identify differences in the protein content of two complex mixtures. For example, the
method can be used to identify protein differences between tumour and normal tissue,
in the same way that 2-D PAGE can be used to address the same question (Section
8.5.1). This method uses two protein ‘tags’ that, whilst being in every other respect
identical, differ slightly in molecular mass; hence one is ‘heavy’ and one is ‘light’.
Both contain (a) a chemical group that reacts with the amino acid cysteine, and (b) a
biotin group. In both molecules these groups are joined by a linker region, but in one
case the linker contains eight hydrogen atoms, in the other, eight deuterium atoms;
one molecule (tag) is thus heavier than the other by 8 Da (see Fig. 9.26). One cell extract
(e.g. from cancer cells) is thus treated with one tag (which binds to cysteine residues
in all the proteins in the extract) and the second tag is used to treat the second extract
(e.g. from normal cells). Both extracts are then treated with trypsin to produce mixtures
of peptides, those peptides that contain cysteine having been ‘tagged’. The two extracts
are then combined and an avidin column used to affinity-purify the labelled peptides
by binding to the biotin moiety. When released from the column this mixture of
labelled peptides will contain pairs of identical peptides (derived from identical
proteins) from the two cell extracts, each pair differing by a mass of 8 Da.
Analysis of this peptide mixture by liquid chromatography–MS will then reveal a
series of peptide mass signals, each one existing as a ‘pair’ of signals separated by eight
mass units. These data will reveal the relative abundance of each peptide in the pair.
Since most proteins present in the two samples originally being compared will be
present at much the same levels, most peptide pairs will have equal signal strengths.
However, for proteins that exist in greater or lesser amounts in one of the extracts,
different signal strengths will be observed for each of the peptides in the pair, reflecting
the relative abundance of this protein in the two samples. Further analysis of either of
these pairs via tandem mass spectrometry will provide some sequence data that should
allow the protein to be identified. ICAT is discussed in more detail in Section 9.6.2.8.5.3 Determining the function of a protein
Successfully applied, the methods described in the preceding section will have pro-
vided the amino acid sequence (or partial sequence) of a protein of interest. The next
step is to identify the function and role of this protein. The first step is invariably to
search the databases of existing protein sequences to find a protein or proteins that
have sequence homology with the protein of interest (the homology method). This is345 8.5 Proteomics and protein function