Page 196 - Veterinary Toxicology, Basic and Clinical Principles, 3rd Edition

VetBooks.ir Chapter 10

Toxicoproteomics in Diagnostic

Toxicology

Christina R. Wilson and Stephen B. Hooser

INTRODUCTION from proteolytic digestion of protein mixtures. Analysis
of these heterogeneous components has proven to be an
During the past decade, advances in genomics research have
analytical challenge due to the large dynamic range of
resulted in the completion of entire genome sequences for
proteins and the microheterogeneity of protein expres-
multiple species. This abundance of genetic information,
sion in biological samples. For instance, serum albumin
compiled in genome sequence databases, has been used to 9
concentrations can range from 35 to 50 3 10 pg/mL
characterize changes in gene expression in response to exter-
and proteins of lower abundance, such as interleukin 6,
nal stimuli, such as toxicants (toxicogenomics). Because
can range in concentration from 0 to 5 pg/mL
genomic data reflects changes in gene expression at the
(Anderson and Anderson, 2002). This is also
mRNA level, DNA sequence databases can be queried to
complicated by protein heterogeneity due to polymorph-
predict what proteins may be present in the cell. However,
isms, alternative mRNA splicing, or variations in post-
this approach is problematic because there is a poor correla-
translational modifications. When analyzing global
tion between mRNA levels and protein concentrations in
proteolytic digests of proteins (i.e., at the peptide level),
cells (Gygi et al., 1999a; Anderson and Seilhamer, 2005).
the sample complexity is compounded further.
Additionally, the biological activity of proteins is controlled
Proteolytic digests of the serum proteome, which could
by posttranslational protein modifications or protein protein
contain as much as 20,000 proteins, can potentially
interactions, both of which cannot be predicted by the geno-
result in 200,000 600,000 peptides (Anderson and
mic data. Therefore, characterizing the entire protein com-
Anderson, 2002; Issaq et al., 2005). Therefore, one can
plement expressed by genes (proteome) in response to
appreciate the analytical challenges encountered when
external stimuli serves as a better indicator of the response
conducting proteomics research in complex biological
of an organism, tissue, or cell to toxic insult. This has given
systems. Attempts to overcome these drawbacks have
rise to the “proteomic” approach to toxicological evaluation.
imposed improvements in sample preparation and
Toxicoproteomics can encompass global analysis of all of
separation methodologies. Multidimensional levels of
the proteins expressed, called “shotgun proteomics,” or can
separation can be achieved when these techniques are
entail analyzing a subset of proteins of interest, often termed
combined affording increased resolution, sensitivity,
“targeted proteomics.” Regardless of the approach,
and accuracy of detection.
proteomics-based research has created an analytical chal-
lenge, requiring sophisticated, high-throughput analytical
techniques, and complex computer algorithms. Two-Dimensional Gel Electrophoresis
Since its inception in 1975 (O’Farrell, 1975), two-
dimensional gel electrophoresis (2-DGE) has become one
ANALYTICAL TOOLS FOR PROTEOMIC
of the most common platforms for separating and profil-
ANALYSES ing complex protein mixtures. This technique is two-
dimensional in that there are two levels of protein
The Proteome and Sample Complexity
separation. The first dimension uses immobilized pH
The initial step in proteome analysis typically involves gradient-isoelectric focusing, which separates proteins
the separation of intact proteins or peptides generated based on differences in net charge (Go ¨rg et al., 2000). In
Veterinary Toxicology. DOI: http://dx.doi.org/10.1016/B978-0-12-811410-0.00010-6
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