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can also be carried out to specifically bind one particular component in a mixture.

Immobilised metal ion affinity columnsare used to enrich phosphopeptides.

9.6.2 Quantitative analysis of complex protein mixtures by mass spectrometry

Proteome analysis (described in Section 8.5) involves the following basic steps:

• run a gel (one-dimensional (1D) or two dimensional (2D)),


• stain,


• scan to identify spots of interest,


• excise gel spots,


• extract and digest proteins,


• mass analyse the resulting peptides,


• search database.

The initial separation of proteins currently relies on gel electrophoresis which has a

number of limitations including the difficulty in analysing all the proteins expressed due

to huge differences in expression levels. Although thousands of proteins can be reprodu-

cibly separated on one 2D gel from approximately 1 mg of tissue/biopsy or biological

fluid, the dynamic range of protein expression can be as high as nineorders of magnitude.

One development that has helped to overcome some of the problems is theisotope-coded

affinity tag(ICAT) strategy for quantifying differential protein expression.

The heavy and light forms of the sulphydryl (thiol-)-specific ICAT reagent (whose

structure is illustrated in Fig. 9.26) are used to derivatise proteins in respective samples

HN

O

O

O O

O

N Y

H

N

H

O

X

XX

X

X

X

X

S X

NH

Biotin Linker Reactive group

Fig. 9.26Structure of the ICAT reagent. The ICAT reagent is in two forms, heavy (eight deuterium atoms) and light

(no deuterium). The reagent has three elements: an affinity tag (biotin), to isolate ICAT-labelled peptides; a

linker in two forms that has stable isotopes incorporated; and a reactive group (Y) with specificity toward thiol

groups or other functional groups in proteins (e.g. SH, NH 2 , COOH). The heavy reagent is D8-ICAT (where X is

deuterium) and light reagent is D0-ICAT (where X is hydrogen). Two protein mixtures representing two different

cell states are treated with the isotopically light and heavy ICAT reagents; an ICAT reagent is covalently attached

to each cysteine residue in every protein. The protein mixtures are combined; proteolysed and ICAT-labelled

peptides are isolated on an avidin column utilising the biotin tag. Peptides are separated by microbore HPLC.

Since each pair of ICAT-labelled peptides is chemically identical they are easily visualised because they co-elute,

with an 8 Da mass difference. The ratios of the original amounts of proteins from the two cell states are strictly

maintained in the peptide fragments. The relative quantification is determined by the ratio of the peptide pairs.

The protein is identified by database searching with the sequence information from tandem MS analysis by

selecting peptides that show differential expression between samples.

392 Mass spectrometric techniques