Page 197 - Veterinary Toxicology, Basic and Clinical Principles, 3rd Edition

164 SECTION | I General

VetBooks.ir the second dimension, proteins are resolved by electro- High Performance Liquid Chromatography
phoresis and separated based on molecular weight.
Attempts to improve protein separations have warranted
Visualization of resolved protein spots is achieved using
universal stains such as Coomassie blue, Coomassie the development of gel-free systems for large-scale analy-
ses. Chromatographic strategies used to fractionate pro-
brilliant blue (SeePico), silver, negative-reversible zinc,
teins and peptides have proven to be a successful
fluorescent, or radioisotope labeling dyes (Go ¨rg et al.,
alternative to 2-DGE. The chromatographic techniques
2000; Lopez et al., 2000; Ong and Pandey, 2001;
commonly employed in proteomics research include affin-
Kuramitsu et al., 2010). If a particular subproteome is
ity chromatography, capillary electrophoresis (CE),
targeted for detection, more specific gel stains can be
hydrophobic interaction chromatography, hydrophilic
used. For example, detection of target proteins
interaction chromatography, ion exchange chromatogra-
can be accomplished using stains containing antibodies
phy (IEX), reversed-phase chromatography (RPC), and
for those proteins of interest or posttranslationally
size exclusion chromatography (SEC) (Goheen and
modified proteins can be visualized using specialized
Gibbins, 2000; Levison, 2003; Goetz et al., 2004; Mahn
stains for phosphorylated and glycosylated proteins
and Asenjo, 2005; Mirzaei and Regnier, 2005; Babu
(Go ¨rg et al., 2004; Vlahou and Fountoulakis, 2005;
et al., 2006; Mondal and Gupta, 2006; Wilson et al.,
Otani et al., 2011).
2008; Di Palma et al., 2011). These modes of chromatog-
After staining, the gel is digitized and protein concen-
raphy fractionate proteins and peptides based on their
trations quantitated using sophisticated 2-DGE image
adsorption and desorption on stationary phase supports
analysis software. Comparative analysis between control
through mobile phase manipulation. On the protein level,
samples and samples of diagnostic interest can be accom-
they are commonly used to prefractionate samples in
plished using these image analyzers to superimpose the
order to simplify complexity prior to analysis by 2-DGE
digitized data from multiple gel runs. However, due to the
or high performance liquid chromatography (HPLC).
laborious nature of this procedure and the lack of gel
Fractionation of proteins using these methods can also be
reproducibility, comparative analysis is often difficult. In
accomplished online using high-throughput HPLC techni-
order to circumvent these limitations, fluorescent dyes,
ques. While this approach has proven to be a successful
known as Cy dyes, have been developed permitting simul-
¨
taneous analysis of two samples on one gel (Unlu ¨ et al., initial step in protein purification, HPLC fractionation of
intact proteins is uncommon in proteomics.
1997; Hamdan and Righetti, 2002). This technique, called
HPLC is, however, the most widely used analytical
two-dimensional differential in-gel electrophoresis (2-D
tool for separating proteolytic digests of complex
DIGE), involves labeling each sample with one of two
protein mixtures. In this approach, all of the proteins
different Cy dye fluorophores, mixing the two samples
in the sample are digested into peptides using a
together, and analyzing them using 2-DGE and
proteolytic enzyme. After digestion, the peptides are
fluorescent-gel imaging.
separated using HPLC with UV detection. When inter-
While the gel-staining techniques used to visualize
faced with a mass spectrometer, the amino acid
and quantitate proteins may vary, in all instances protein
sequences of these peptides can be determined and
identification is commonly accomplished using tandem
their respective parent proteins identified using bioin-
mass spectrometry (MS/MS). Excised protein spots are
formatics software.
digested into peptides using proteolytic enzymes and sub-
Large-scale analysis of proteolytic digests can com-
jected, offline, to MS/MS analysis. The peptide mass fin-
promise the resolving power of HPLC when only one
gerprints generated and peptide sequence data are then
chromatographic fractionation technique is used (Davis
compared to theoretical peptide masses in protein or
and Giddings, 1985a,b). Therefore, orthogonal approaches
genome sequence databases using specialized bioinfor-
using multidimensional chromatographic separations are
matics algorithms.
preferred, affording greater peptide resolution and accu-
Despite the fact that improvements in 2-DGE technol-
racy of detection. The most widely used high-throughput
ogy have enabled high resolution of separated proteins
separation technique is the coupling of IEX and RPC with
and enhanced protein identification, some intrinsic pro-
MS instrumentation. Other examples of multidimensional
blems remain. Limited throughput capabilities, inability
HPLC/MS techniques can also include RPC/CE chroma-
to measure low and high molecular mass proteins, intergel
tography, affinity chromatography/RP chromatography,
variability and inefficient detection of basic and hydro-
SEC/IEX/RPC, and SEC/RPC/CE (Issaq et al., 2005;
phobic proteins are still inherent limitations of this proteo-
Zhang et al., 2010a).
mics platform. Regardless of these limitations, 2-DGE is
HPLC has also been used for quantitative analysis of
still a powerful tool for the separation of intact proteins
protein expression. Relative quantitation of proteins by
and is the most widely used technique in comparative tox-
HPLC is based on the theory that the concentration of the
icoproteomic analyses.