Toxicoproteomics is the use of global protein expression technologies to better
understand environmental and genetic factors, both in episodes of acute exposure
to toxicants and in the long-term development of disease. Integrating transcript,
protein, and toxicology data is a major objective of the field of toxicogenomics.
Toward that goal, the NCT is pursuing a strategy of conducting parallel DNA
microarray and proteomic analyses on the same tissues from each toxicogenomics
study. This parallel approach combines the high level of gene discovery of microarray
analysis with the utility of proteomics in exploiting post-translational modifications
of target proteins affected by toxicants to identify biomarkers.
The Proteomics Program within the NCT consists of an intramural program, a
proteomics resource contract, extramural awards in proteomics, and Small Business
Innovation Research awards. The close association of the Proteomics Program
with the NIEHS Microarray Center and interactions with the National Toxicology
Program also provide unique opportunities for integrating genomics, proteomics,
and toxicology into joint studies.
Methods
A primary goal of the intramural program is the discovery of biomarkers in
organs and serum that reflect toxicant exposure or environmental disease, using
both established and emerging proteomics technologies. For example, two-dimensional
gel electrophoresis and MS/MS are being used for global protein separation and
protein identification. SELDI is being investigated for potential use in serum
biomarker development and classification of control and toxicant-exposed experimental
animals. Strong capabilities have been built in capillary and nanoscale LC separations
and in the use of MS for the identification and fine structure characterization
of protein samples digested by enzymes. There is also an emphasis on LC/MS/MS
approaches to differential protein expression, such as the use of isotope-coded
affinity tags and characterization of post-translational modifications. Additional
capabilities include MALDI/TOF/MS, MALDI/TOF/TOF MS/MS, nanoscale LC/ESI/Q-Tof
MS/MS, N- and C-terminal protein microsequencing, several capillary HPLCs, a
proteomics laboratory information management system, and a National Center for
Biotechnology Information database with search capabilities that is housed on
campus. Efforts in this area are coordinated by two research groups, the NCT
Proteomics Group and the Mass Spectrometry Group in the NIEHS Laboratory of
Structural Biology.
Investigations
Researchers in the Proteomics Program are particularly interested in investigating
protein phosphorylation as a key post-translational event in pathway signaling
and in protein degradation. New fluorescent phosphorylation sensor dyes, IMAC
techniques, and site-specific phosphopeptide biotinylation are three new procedures
being used to detect and capture large numbers of phosphorylated proteins and
peptides simultaneously. Further, MALDI/MS and LC/MS/MS techniques are being
used to identify changes in phosphopeptides altered by exposure to toxicants.
Investigation of cellular and subcellular proteomes is another area in which
NCT researchers are exploiting toxicoproteomics. Although most tissues are amenable
to RNA extraction for generation of cDNA and hybridization to thousands of genes
arrayed on DNA chips, protein analysis often involves reductive procedures whereby
the complex structure of organs is lost. NCT researchers are retaining a certain
level of organization by isolating specialized cell types within the organ and
fractionating organs into subcellular components and organelles. Because liver
tissue can be analyzed whole or as subcellular fractions, these techniques are
being used to investigate liver toxicity.
Although the liver consists primarily of hepatic parenchymal cells, several
nonparenchymal cells--including stellate cells, pit cells, Kupffer cells, biliary
cells, and endothelial cells--also are important for liver function. These cells
may be targets for toxicity and may increase in number in some toxic and pathologic
conditions to significantly alter the hepatic protein profile. Isolation of
these cells in their resting and active states may reveal protein profiles contributing
to liver toxicity that would have been very difficult to observe in a mixed
population of cells dominated by hepatocytes.
Fractionation of subcellular organelles is a means to enrich for structurally
meaningful subcellular units of the liver in a way that is not possible by protein
analysis of whole liver homogenates or even by RNA isolation and transcript
analysis. Enriched fractions of nuclei, mitochondria, endoplasmic reticulum,
plasma membranes, and cytosol can provide insight into the potential effects
of a toxicant on a particular site within subcellular structures or upon protein
trafficking or signaling pathways.
Proteomics platforms appear particularly well suited for biomarker development
in blood because the appearance of new polypeptides in the serum proteome may
reveal early-stage disease and organ toxicity. In the biofluids produced by
each organ, serum and plasma proteomes of blood can uniquely reveal signs of
specific organ toxicity or pathology from the peptides and proteins passively
leaked or actively secreted during dysfunction. In addition, blood carries the
nutrients, intermediary metabolites, amino acids, peptides, proteins, and enzymes
necessary for organ health, adaptation, and repair of organ toxicity. Such blood-transported
biomaterials may arise from other body tissues as well as from alimentary absorption.
Analysis of serum or plasma alone presents a challenge in separation and detection
of informative proteins due to the adundance of other serum proteins such as
albumin, immunoglobulins, and transferrin. NCT researchers are using immunoaffinity
and ligand-affinity columns to remove such numerous and noninformative serum
constituents--which comprise almost 90% of serum--to allow for separation and
identification of the remaining 10% of informative serum proteins.
Strategies
NCT researchers in the Proteomics Program are in the process of developing
and refining strategies for integrating toxicogenomics and toxicoproteomics
data.
image credit: B. Alex Merrick, Matt Ray/EHP |
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