Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 5 Proteins, Proteomics, and the Dysproteinemias
3 . Polyacrylamide Gel Electrophoresis and
Isoelectric Focusing
The most widely used support medium for protein electrophoresis
outside the diagnostic laboratory is polyacrylamide
gel (PAGE). The use of this medium brings a further factor
to the electrophoretic separation of protein. During the
polymerization of acrylamide to form the gel used in electrophoresis,
the proportion of cross-links between polymer
chains can be controlled, and the gel forms a molecular
sieve that slows the migration of proteins depending on size.
In its original version of discontinuous polyacrylamide gel
electrophoresis ( Davis, 1964 ), a strategic use of different
buffer systems in the gel, in the sample, and in running buffer
caused the proteins in the sample to focus into a sharp
band before entering the gel. Once in the gel, separation was
based on a balance of mass and charge on the protein. The
most widely used modification of this system is to pretreat
the proteins by heating in a solution of detergent (sodium
dodecyl sulphate, SDS) and a reducing agent such as β -
mercaptoethanol. These have the effect of separating any
subunits held together by disulphide bonds and coating all
the proteins with negative charge so that separation, with the
same detergent also in the gel and buffers, is based on size
alone as all protein will move to the anode because of their
negative charge. This is the SDS-PAGE system introduced
by Laemmli ( Laemmli, 1970 ). Separation of serum protein
on SDS-PAGE increases the complexity for interpretation of
the electrophoretogram. The proteins are no longer grouped
in the familiar globulin regions but are in a series of bands
defined by relative molecular mass (M r ). The treatment and
breakdown of complex proteins into their component subunits
complicate interpretation. The high abundance of just a
few of the proteins, such as albumin and the immunoglobulins,
causes further difficulty in interpretation. Added to this
are the more technically demanding methods required for
SDS-PAGE such that this method is largely confined to the
research laboratory. Nevertheless, separation of serum protein
by SDS-PAGE has revealed disease-related changes in
protein bands ( Fagliari et al. , 1998 ; Kiral et al. , 2004 ), but
there has not been a widespread application of the method in
diagnostic biochemistry.
A further separation technique for electrophoretic
fractionation of protein mixtures, introduced in the 1970s
( Righetti and Drysdale, 1971 ), is isoelectric focusing
(IEF). This technique, which can be performed in agarose
or polyacrylamide gels, differs from other forms of electrophoresis
by separating the proteins solely on the basis
of their charge. The presence of special reagents, called
ampholytes, in the buffer creates a pH gradient once an
electric voltage is set up across the gel. Proteins in the gel
move because of their relative charge, but once they reach
their isoelectric point (pI) on the pH gradient, they become
stationary, as they now have zero charge. Thus, an acidic
protein with a negative charge will move toward the anode,
but as it moves down the pH gradient the protein becomes
less charged until it reaches the point where it has no net
charge and it is “ focused ” at their pI. This method has a
high resolution and can separate protein isoforms that have
only slight charge differences caused, for instance, by glycosylation
or phosphorylation of proteins. This greatly
increases the potential number of bands that can be seen
on IEF gels, but the method has not been adopted by diagnostic
clinical biochemistry laboratories for separation of
serum proteins, possibly because of this great complexity.
However, IEF has been used in examination of enzyme
isoforms ( Eckersall and Nash, 1983 ) and can be used to
identify microheterogeneity in specific serum proteins, for
instance, being able to differentiate multiple forms of AGP
that are caused by different degrees of glycosylation ( Itoh
et al., 1993a ; Yoshida et al. , 1997 ).
4 . Proteomics
Protein analysis is currently going through rapid evolution
that could impact the veterinary diagnostic laboratory in
the not-too-distant future and is being driven by advances
in proteomics. Technological developments in different
disciplines have converged to produce an approach to the
separation, identification, and quantification of individual
proteins within a complex mixture. The objective of a
proteomic investigation is to be able to identify all proteins
in a tissue or fluid and to detect even small changes
taking place in its composition. Although this goal is
still beyond the reach of all but the best-funded research
laboratories, it is probable that proteomic techniques will
eventually be used in diagnosis of disease. Analysis of
serum or plasma protein will be at the forefront of these
advances. It has been suggested that the human plasma
proteome could be used to detect virtually all pathological
processes because every diseased tissue is in contact
with the circulation and interchanges material with plasma
( Anderson and Anderson, 2002 ). As many as 1175 distinct
gene products have been reported in human plasma by a
combination of methods ( Anderson et al. , 2004 ), whereas
289 proteins have been directly detected. However, only
117 of these have been registered in the Untied States
by the Food and Drug Administration under the Clinical
Laboratory Improvement Amendment for use in diagnostic
investigation of plasma ( Anderson and Anderson, 2002 ).
Investigation of the diagnostic potential of animal serum or
plasma proteomes is at a much earlier stage, but it has the
potential to yield many novel diagnostic applications.
a . Two-Dimension Gel Electrophoresis
The new science of proteomics ( James, 1997 ) initially
developed from methods in which the electrophoretic
techniques of IEF and SDS-PAGE were combined into
two-dimensional electrophoresis (2DE) ( O’Farrell, 1975 ).
Combination of these methods leads to a protein map,