18.2 Proteomics

279

Fig. 18.2 A

two-dimensional gel after

staining

be stained and the gel scanned with a densitometer; the spot density is then propor-

tional to protein abundance. There are some caveats: Membrane proteins with more

than two transmembrane sequences are poorly recovered by the technique; if Superscript 35³⁵S

met/cys is used, one should note that not all proteins contain the same number of

met and cys (but this number is only very weakly correlated with molecular weight);

autoradiography may underestimate the density of weak spots, due to low-intensity

reciprocity failure of the photographic (silver halide) film used to record the presence

of the radionucleides; the commonly used Coomassie blue does not stain all proteins

evenly, although the unevenness appears to be random and hence should not impose

any systematic distortion on the data; rare proteins may not be detected at all; sev-

eral abundant proteins clustered close together may not be distinguishable from each

other; and very small and very large proteins, and those with isoelectric points (pI) at

the extremes of the pH range, will not be properly separated. The molecular weight

and isoelectric point ranges are limited by practical considerations. Typical ranges

are 15 000 less than upper M Subscript normal r Baseline less than 90 00015 000 < Mr < 90 000 and 3 less than< pI less than< 8. Hence, the mostly basic (pI typically in

the range 10–14) 50–70 ribosomal proteins will not be captured, as a notable example

(on the other hand, these proteins are not supposed to vary much from cell to cell,

regardless of conditions, since they are essential proteins for all cells; hence, they

are not considered to be especially characteristic of a particular cell or metabolic

state). Figure 18.2 shows a typical result. Such images are obvious candidates for

refinement using maximum entropy techniques (Sect. 13.5).

Problem.

Write

a

computer

program

for

reconstructing

a

poor-quality

two-dimensional gel electrophoretogram using the principle of maximum entropy.

18.2.2

Column Chromatography

The principle of this method is to functionalize a stationary solid phase (granules of

silica, for example) packed in a column and pass the sample (suspended or dissolved