- observe the response to extracellular stimuli such as hormones or cytokines;
- compare pathogenic and non-pathogenic bacterial strains;
- compare serum protein profiles from healthy individuals and Alzheimer or cancer
patients to detect proteins, produced in the serum of patients, which can then be
developed as diagnostic markers for diseases (e.g. by setting up an enzyme-linked
immunosorbent assay (ELISA) to measure the specific protein).
As a typical example, a research group studying the toxic effect of drugs on the
liver can compare the 2-D gel patterns from their ‘damaged’ livers with the normal
liver 2-D reference map, thus identifying protein changes that occur as a result of
drug treatment.
The sheer complexity and amount of data available from 2-D gel patterns is
daunting, but fortunately there is a range of commercial 2-D gel analysis software,
compatible with personal computer workstations, which can provide both qualitative
and quantitative information from gel patterns, and can also compare patterns
between two different 2-D gels (see below). This has allowed the construction of a
range of databases of quantitative protein expression in a range of tissue and cell
types. For example, an extensive series of 2-DE databases, known as SWISS-2D PAGE,
is maintained at Geneva University Hospital and is accessible via the World Wide Web
at >http://au.expasy.org/ch2d/>. This facility therefore allows an individual labora-
tory to compare their own 2-D protein database with that in another laboratory.
The comparison of two gel patterns is made by using any one of a number of
software packages designed for this purpose. One of the more interesting approaches
to comparing gel patterns is the use of the Flicker program, which is available on
the Web at >http://open2dprot.sourceforge.net/Flicker>. This program superimposes
the two 2-D patterns to be compared and then alternately, and rapidly, displays one
pattern and then the other. Spots that appear on both gel patterns (the majority) will
be seen as fixed spots, but a spot that appears on one gel and not the other will seen to
be flashing (hence ‘flicker’). When one has compared two 2-DE patterns and identified
any proteins spot(s) of interest, it is then necessary to identify each specific protein. In
the majority of cases this is done by peptide mass-fingerprinting. The spot of interest
is cut out of the gel and incubated in a solution of the proteolytic enzyme trypsin,
which cleaves the protein C-terminal to each arginine and lysine residue. In this way
the protein is reduced to a set of peptides. This collection of peptides is then analysed
by MALDI-MS (see Section 9.3.8) to give an accurate mass measurement for each of
the peptides in the sample. This set of masses, derived from the tryptic digestion of the
protein, is highly diagnostic for this protein, as no other protein would give the same
set of peptide masses (fingerprint). Using Web-based programs such as Mascot or
Protein Prospector this experimentally derived peptide mass-fingerprint is compared
with databases of tryptic peptide mass-fingerprints generated from sequences of
known proteins (or predicted sequences deduced from nucleotide sequences). If a
match is found with a fingerprint from the database then the protein will be identified.
However, sometimes results from peptide mass-fingerprinting can be ambiguous.
In this case it is necessary to obtain some partial amino acid sequence data from one
of the peptides. This is done by tandem mass spectrometry (MS/MS; Section 9.5),342 Protein structure, purification, characterisation and function analysis