280

18

Transcriptomics and Proteomics

in the liquid mobile phase) through it (cf. Sect. 23.8.1). The functionalization is such

that the proteins of interest are bound to the granules, and everything else passes

through. A change in the liquid phase composition then releases the bound proteins.

Better separations can be achieved by “multidimensional” liquid chromatography

(MDLC), in which a cation exchange column (for example) is followed by a reverse

phase column. The number of “dimensions” can obviously be further increased.

Usually, the technique is used to prepurify a sample, but, in principle, using differen-

tial elution (i.e., many proteins of interest are bound and then released sequentially

by slowly increasing pH or polarity of the liquid), high-resolution analytical sep-

arations may also be accomplished. Miniaturization (nano-liquid chromatography)

offers promise in this regard. The output from the chromatography may be fed directly

into a mass spectrometer (MS).

In MudPIT (multidimensional protein identification technology), the proteins are

first denatured and their cysteines reduced and alkylated, and then digested with

a protease. Following acidification, the sample is then passed through a strong

cation exchange chromatographic column, followed by reverse phase chromatog-

raphy. Eluted peptides are introduced into a mass spectrometer (typically a tandem

(MS/MS) instrument) for identification (see Sect. 18.3).

18.2.3

Other Kinds of Electrophoresis

Free fluid electrophoresis (FFE) is distinguished from chromatography in that there is

no stationary phase (i.e., no transport of analytes through a solid matrix such as a gel).

The separation medium and the analytes are carried between electrodes, arranged

such that the electric field is orthogonal to the flow of the separation medium. ¹¹

18.3

Protein Identification

Two-dimensional gel electrophoresis is very convenient since it creates a physical

map of the cell’s proteins inupper M Subscript rMr–i.e.p. space, from which the proteins at given coor-

dinates can actually be cut out and analysed. Hence, it is possible to apply Edman

sequencing, ¹² at least to the more abundant proteins, or Stark C-terminal degradation.

The most widely applied technique is based on MS, however. ¹³ It is capable of much

11 See Patel and Weber (2003) for a review.

12 The N-terminal of the protein is derivatized with phenylisothiocyanate to form a phenylthiocar-

bamate peptide, and the first amino acid is cleaved by strong acid resulting in its anilothiazolinone

derivative plus the protein minus its first N-terminal amino acid. The anilothiazolinone derivative is

converted to the stabler phenylthiohydantoin for subsequent high-performance liquid chromatog-

raphy (HPLC) identification.

13 See Bell et al. (2009) for efforts to overcome errors in mass spectrometry-based proteomics.