18.2 Proteomics

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18.2

Proteomics

The proteome can be accessed directly by measuring the expression levels, not of the

mRNA transcripts but of the proteins into which they are translated. Not surprisingly,

in the relatively few cases for which comparative data for both the transcriptome and

proteome have been obtained, the amounts of the RNAs and corresponding proteins

may be very different, even if all the different proteins derived from the same RNA

are grouped together—translation is an important arena for regulating protein syn-

thesis. Before this became apparent, transcriptomics acquired importance because

technically it is much easier to obtain the transcriptome using a microarray than

it is to obtain the proteome using laborious two-dimensional gel electrophoresis

(Sect. 18.2.1), for example. It was hoped that the transcriptome would be a reason-

ably faithful mirror of the proteome. This is, however, definitely not the case in

general; there is no presently discernible unique relationship between the abundance

of mRNA and the abundance of the corresponding protein. Hence, the transcriptome

has lost some of its importance; it is “merely” an intermediate stage and does not

contribute directly to phenotype in the way that the proteome does. Furthermore, the

transcriptome contains no information about the very numerous post-translational

modifications of proteins. On the other hand, to understand the relation between

transcriptome and proteome would be a considerable advance in understanding the

overall mechanism of the living cell. Given that both transcriptome and proteome

spaces each have a high dimensionality, deducing a relation between trajectories in

each is still a rather forlorn hope.

The first step in proteomics proper is to separate all of the expressed proteins from

each other such that they can be individually quantified (i.e., characterized by type

and number). Prior to that, however, the ensemble of proteins has to be separated

from the rest of the cellular components. ⁹ Cells are lysed, proteins are solubilized,

and cellular debris is centrifuged down. Nucleic acids and lipids are removed and

sometimes very abundant proteins (such as albumin from serum). A subset of proteins

may be labelled at this stage, to assist later identification.

A particularly useful form of labelling is to briefly (for 30–40 min) feed the liv-

ing cells with radioactive amino acids (Superscript 35³⁵S-cysteine and methionine are suitable),

followed by an abundance of nonradioactive amino acids (pulse radiolabelling). The

degree of incorporation of radioactivity into the proteins is then proportional to the

net rate of synthesis (i.e., biosynthesis rate minus degradation rate).

The two main techniques for separating the proteins in this complex mixture

(which is likely to contain several hundred to several thousand different proteins) are

the following:

1. Two-dimensional gel electrophoresis (2DGE);

2. Enzymatic proteolysis into shorter peptides followed by column chromatography.

Trypsin is usually used as the proteolytic enzyme (protease) since it cuts at well-

defined positions (lysines).

9 See Wis´niewski (2009).