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18

Transcriptomics and Proteomics

The protein mixture may be pretreated (prefractionated), using chromatography or

electrophoresis, before proceeding to the separation step, in order to selectively enrich

it with certain types of proteins.

Problem. List and discuss the differences between mRNA and protein abundances.

18.2.1

Two-Dimensional Gel Electrophoresis

In order to understand the principles of protein separation by 2DGE, let us first recall

some of the physicochemical attributes of proteins. Two important ones are

1. Molecular weight (relative molecular mass upper M Subscript normal rMr);

2. Net electrostatic chargeupper ZZ [as a function of pH—the pH at whichupper Z equals 0Z = 0 is impor-

tant as a characteristic parameter, known as the isoelectric point (i.e.p.), or pI, or

point of zero charge (p.z.c.)].

Both can be calculated from the amino acid sequence (assuming no posttranslational

modifications), provided upper M Subscript normal rMr and upper ZZ of the individual amino acids are known. upper M Subscript normal rMr is

easy; to calculate upper ZZ, one has to make the quite reliable assumption that all of the

ionizable residues are on the protein surface. The calculation is not quite as simple

as adding up all the surface charges, since they mutually affect each other (cf. the

surface of a silicate mineral: not every hydroxyl group is ionized, even at extremely

low pH). ¹⁰

The technique itself was developed by Klose and, independently, by O’Farrell in

1975. The concept depends on the fact that separation by isoelectric point (i.e.p.) is

insufficient to separate such a large number of proteins, many of whose i.e.p.s are

clustered together. Equally, there are many proteins with similar molecular masses.

By applying the two techniques sequentially, however, they can be separated, espe-

cially if large (30 times 4030 × 40 cm) gels are used.

Proteins in the crude cell extract are dispersed in an aqueous medium containing

the anionic detergent sodium dodecyl sulphate (SDS), which breaks all noncovalent

bonds (i.e., subunits are dissociated, and probably denatured too); the first separation

takes place according to the i.e.p. by electrophoresis on a gel along which a pH

gradient has been established; the partly separated proteins are then transferred to

a second, polyacrylamide, gel within which separation is effected according to size

(i.e., molecular weight if all proteins are assumed to have the same density).

If the cells have been pulse radiolabelled prior to making the extract, then the final

gel can be scanned autoradiographically and the density of each spot is proportional

to the net rate of protein synthesis. Alternatively (or in parallel), the proteins can

10 Linderstrøm-Lang worked out a method of taking these correlations into account; his formula

works practically as well as more sophisticated approaches (including explicit numerical simulation

by Brownian dynamics; cf. Madura et al. 1994) and is much simpler and more convenient to calculate

(see Ramsden et al. (1995) for an application example).