74
techniques, which identifi es both the amino acid sequences of proteins and their
posttranslational appendages. This approach is combined with database search
algorithms to sequence and characterize individual proteins.
2D Gel Electrophoresis
2D gel electrophoresis (2DGE) offers the highest resolution separations available
for protein components of cells when gels of suffi cient size are used. Proteins are
separated in the fi rst dimension on the basis of their charge and in the second dimen-
sion on the basis of their molecular mass. 2DGE is still the workhorse for obtaining
protein expression patterns in cells. In high-format mode, it can produce gels con-
taining up to 10,000 distinct proteins and peptide spots. The major problem with
this technique is that most of the spots cannot be sequenced as they are beyond the
capacity of current high-sensitivity sequencers. Standard format 2D gels yield up to
2,000 spots and are easy to sequence. During 2D PAGE (polyacrylamide gel elec-
trophoresis), the proteins are separated in two dimensions (by isoelectric focusing
and mass) and a pattern is achieved that places each of the 2,000 proteins of the cell
at a grid reference point. By reference to the databases, individual proteins on the
map can be identifi ed as the product of genes that have been sequenced.
While comparing different samples, controlling the position of the protein spots
can be critical and is completely dependent upon the fi delity of the isoelectric focus-
ing fi rst dimension and the molecular weight separating gel slab of the second
dimension. Differences between the test samples are determined by quantifying the
ratios of spot intensities in independent 2D gels and techniques such as mass spec-
trometry (MS) can then be used to help identify the proteins through peptide mass
fi ngerprinting or direct sequencing. A number of variations in the basic 2DGE tech-
nology have enhanced separation of protein components in a sample.
Although 2DGE is the most widely used tool for separating proteins in expres-
sion proteomics, it is not without its limitations. Challenges faced when utilizing
this technology are co-migration of proteins, systematic exclusion of highly hydro-
phobic molecules, and problems with detecting very acidic, very basic, very small,
very large, or low abundance proteins. To meet the demands of protein separation,
companies are developing new technologies that appear to be inexpensive and reli-
able, generate high-resolution protein separation and yield good visual detection of
subtle differences. Competing emerging technologies such as capillary electropho-
resis, capillary electrochromatography and ultra-HPLC (high performance liquid
chromatography) are beginning to replace 2DGE.
Mass Spectrometry
Mass spectrometry (MS) is the measurement of molecular mass. A mass spectrom-
eter consists of three essential parts: (1) an ionization source with conversion of
molecules into gas-phase ions; (2) a mass analyzer to separate individual mass to
charge rations (m/z); and (3) an ion detector. Several variants of MS are described
in the following sections ( see Fig. 2.7 ).
2 Molecular Diagnostics in Personalized Medicine