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

V. Methodology
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pI 4.5 5 6 8 4.5 5 6 8 4.5 5 6 8
FIGURE 5-4 Two-dimensional electrophoresis of serum protein from (a) horse, (b) cow postcolostrum, (c) cow
precolostrum, (d) sheep, (e) cat, and (f) dog. Labeled proteins are 1: albumin, 2: transferrin, 3: IgG heavy chain,
and 4: IgG light chain. Gels were run on an IPG gradient from pH 4-10 (nonlinear) and then on SDS-PAGE.
Gels courtesy of Ingrid Miller, Institute of Medical Chemistry, Department of Natural Sciences, University of
Veterinary Medicine, Vienna, Austria.
where a protein mixture is separated horizontally by charge
(IEF) and vertically by molecular mass (SDS-PAGE) yielding
a two-dimensional map with each protein present as a
single spot. An innovation that meant that these protein
maps were much more reproducible was the introduction
of immobilized pH gradients for use in the IEF step ( Gorg
et al. , 2000, 2004 ). In 2DE, the protein sample is subjected
to IEF in a gel strip containing the immobilized pH gradient,
and then the strip with the focused protein is placed
on the top of an SDS-PAGE gel. After electrophoresis,
the separated proteins are stained, using either Coomassie
blue or the more sensitive silver or fluorescent stains. The
amount of data generated by a 2DE gel can be vast, and a
computer program is required to handle the analysis.
The serum proteomes of a number of domestic animals
are illustrated in Figure 5-4 . It is noticeable that albumin is the
most abundant protein in adult serum and that the IgG spots
are missing in serum from a precolostral calf ( Fig. 5-4c ).
The serum proteomes of cattle and horse have been more
fully determined with 30 and 50 proteins identified,
respectively ( Miller et al. , 2004 ; Wait et al. , 2002 ).
Identification of protein spots following 2DE was originally
performed with antibody detection, specific stains
(e.g., for lipoprotein), or by comparison to the proteins
of other species. A further advance that greatly facilitated
proteomic research was the use of mass spectrometry to
identify protein spots on gels.
b . Mass Spectrometry for Protein Identification
Mass spectrometry has been used for many years in investigations
to measure the mass of molecules to a high degree of
accuracy, but for a long time it was restricted to low-molecular-weight
compounds. In the 1980s and 1990s, methods
were introduced to determine the mass of larger molecules
such as peptides and smaller protein. This was achieved
with the introduction of electro spray ionization (ESI) ( Fenn
et al. , 1989 ) and matrix-assisted laser desorption/ionization
(MALDI) ( Karas and Hillenkamp, 1988 ). These methods
are central to alternative approaches to identify the
protein on 2DE gels and have accelerated the development