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18

Transcriptomics and Proteomics

18.4

Isotope-Coded Affinity Tags

Isotope-coded affinity tags (ICATs) ¹⁵ are particularly useful for comparing the

expression levels of proteins in samples from two different sources (e.g., cells before

and after treatment with a chemical). It is a way of reducing the variety (number

of proteins that have to be separated) of a complex mixture. Proteins from the two

sources are reacted with light and heavy ICAT reagents in the presence of a reduc-

ing agent. The reagents comprise a biotin moiety, a sulfhydryl-specific iodoacetate

moiety, and a linker that carries eight Superscript 1¹H (light) or squared²H (heavy) atoms. They specifi-

cally tag cysteinyl residues on the proteins. The two batches are then mixed and the

proteins cleaved using trypsin. The fragments, only about a fifth of which contain

cysteine, can be readily separated by chromatography on an avidin affinity column

(which binds to the biotin), and finally analysed by MS. Singly charged peptides

of identical sequences from the two sources are easily recognized as pairs differing

by eight atomic mass units. Differences in their expression levels can be sensitively

compared and normalized to correct for differences in overall protein content.

Many other affinity enrichment techniques can be imagined, tailored according to

the proteins of interest; for example, lectins can be used to make a column selectively

capturing glycoproteins.

18.5

Protein Microarrays

Generic aspects of microarrays have already been covered in Sect. 18.1. Protein

microarrays allow the simultaneous assessment of expression levels for thousands of

genes across various treatment conditions and time. The main difference compared

with nucleic acid arrays is the difficulty and expense of placing thousands of protein

capture agents on the array. Since capture does not depend on simple hybridization,

but on a certain arrangement of amino acids in three-dimensional space, complete

receptor proteins such as antibodies have to be used, and then there is the danger

that their conformation is altered by immobilization on the chip surface. ¹⁶ It may

be possible to exploit the advantages of nucleic acid immobilization (especially the

convenient photofabrication method) by using aptamers—oligonucleotides binding

15 Developed by Aebersold (see Gygi et al. 1999.).

16 As an alternative way to prepare oligopeptide receptors, the phage display technique invented

by Dyax is very useful. The gene for the coat protein expressed abundantly on the surface of

a bacteriophage virus is modified by adding a short sequence coding for an oligopeptide to one

end. Typically, a large number (tilde 10 Superscript 9∼10⁹) of random oligonucleotides is synthesized and incorporated

(one per phage) into the virus gene. The phages are then allowed to multiply by infecting a host

bacterium; the random peptide is expressed in abundance on the coat of the phage along with the

regular coat protein. The phage population is then exposed to an immobilized target (e.g., a protein).

Any phage (a single one suffices) whose peptide interacts with the target during this screening is

retained and recovered, and then multiplied ad libitum in bacteria.