General and Comparative Endocrinology 164 (2009) 135141

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General and Comparative Endocrinology

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Towards functional genomics in sh using quantitative proteomics

Christopher J. Martyniuk *, Nancy D. Denslow
Center for Environmental and Human Toxicology, Department of Physiological Sciences, University of Florida, P.O. Box 110885, Gainesville, FL 32611-0885, USA

a r t i c l e i n f o a b s t r a c t

Article history: Microarray and gene expression analysis have been key in our understanding of molecular pathways
Received 25 August 2008 underlying physiological responses. Arguably, a large number of microarray based studies in sh have
Revised 26 January 2009 examined steroid nuclear receptor signaling (e.g., estrogens, androgens) in the context of both physiology
Accepted 26 January 2009
and toxicology. Following close behind the advances in gene expression analysis, novel proteomic tools
Available online 5 February 2009
are available that have been under utilized in sh endocrinology studies. Quantitative proteomic
approaches include both gel based (e.g., 2D gel electrophoresis, 2-D Fluorescence Difference Gel Electro-
Keywords:
phoresis; DIGE) and non-gel based methods that can be separated further into labeling approaches such
Quantitative proteomics
Fathead minnows
as stable isotope labeling (SILAC), isotope coded afnity tags (ICAT), and isobaric tagging (iTRAQ) and
17b-trenbolone label-free approaches (e.g., spectral counting and absolute quantitation). This review summarizes quan-
Genomics titative proteomic approaches and describes a successful application of iTRAQ to study changes in the
liver proteome in fathead minnows in response to the androgen, 17b-trenbolone. The challenge remains
to integrate molecular datasets in such a manner as to be able to consider temporal effects and complex
regulation at the level of the genome and proteome.
2009 Elsevier Inc. All rights reserved.

1. Functional genomics and proteomics
Genomic approaches using microarrays have increased our
understanding of molecular pathways regulated in sh tissues by
hormones such as reproductive steroids (Larkin et al., 2007; Moens
et al., 2007; Santos et al., 2007; Hoffmann et al., 2008; Marlatt
et al., 2008). With the vast increase in genomic information, it
becomes increasingly important to provide a functional compo-
nent to the genomic changes observed using methods such as gene
knockdown or over-expression studies, mapping regulatory gene
networks, or anchoring molecular pathways to physiological or
behavioral phenotypes. The concept of functional genomics also in-
cludes studying how genomic changes correlate to changes at the
protein level as well as to protein activation/deactivation via phos-
phorylation and phosphatase activities.
In general, proteomics is the study of the entire complement of
proteins expressed spatially and temporally in an organism,
including protein variants and post-translational modications,
and the characterization of proteinprotein interactions. The pro-
teome is magnitudes larger than the genome, and some genes,
for example neurexins, can have upwards of 1000 protein isoforms
(Ullrich et al., 1995). Due to the complexity of the proteome, sep-
aration of protein mixtures becomes important for identifying
and quantifying proteins.
Along with 2D gel electrophoresis, multidimensional separation
techniques using high performance liquid chromatography have
facilitated the large scale study of the proteome. Multidimensional
Protein Identication Technology (MudPit) separates tryptic pep-
tides obtained from total protein via two high performance liquid
chromatography (HPLC) approaches, strong cation exchange (SCX)
back-to-back with reversed-phase (RP), to reduce the total complex-
ity of the peptide preparation before MS/MS (Washburn et al., 2001).
Combined fractional diagonal chromatography or COFRADICTM
(Gevaert et al., 2002) isolates peptides rst by RP-HPLC. Then, eluted
peptides are modied by chemical or enzymatic means for specic
amino acids or functional groups (e.g., methionine or phospho-
groups) and separated again by RP-HPLC. The modied peptides
exhibit different retention times during RP-HPLC than the original
unmodied peptides, sorting the complex mixture. Although these
methods act to reduce the complexity of the proteome for study,
the proteomic coverage compared to the genome still remains com-
paratively small. For example, microarray platforms are becoming
increasingly dense, containing 2040 thousand unique elements
on a single slide. In comparison, the proteomic coverage obtained
by 2D gel electrophoresis is approximately 2000 proteins, amount-
ing to 2% of the total proteome and it currently remains a signicant
challenge to increase the proteomic coverage.
2. Gel-based separation for protein quantitation

* Corresponding author. Fax: +1 352 392 4707. Quantitative proteomic approaches can be separated into both
E-mail address: cmartyni@u.edu (C.J. Martyniuk). gel based and non-gel based methods (Fig. 1). The more classical

0016-6480/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2009.01.023
136 C.J. Martyniuk, N.D. Denslow / General and Comparative Endocrinology 164 (2009) 135141
Fig. 1. General classication scheme of label and label-free quantitation techniques
available to study teleost proteomes.
gel based methods include 2D gel electrophoresis, with proteins
being separated in the rst dimension by isoelectric point followed
by the second dimension by protein mass on a sodium dodecyl sul-
fate polyacrylamide gel. Technical experience is a signicant factor
in the successful application of 2D gel electrophoresis and an
obstacle to overcome is gel to gel variation due to running time
and separation, which increases the difculty in correlating spots
across gels. In addition, the co-migration of multiple proteins in
overlapping spots and proteins with low signals are difculties de-
spite assistance from automated software programs.
To reduce gel to gel variation, difference gel electrophoresis
(DIGE) utilizes uorophores to measure relative protein abundance
within a single gel (Unl et al., 1997). Similar to two dye micro-
array experiments, cyanine (Cy) 3 and 5 dyes are commonly used
to label both control and treatment protein groups. In addition, a
third dye (Cy2) is used to label an internal protein standard which
is added into each control and treatment protein mix before iso-
electric focusing and electrophoresis separation. This allows the re-
searcher to compare protein changes by standardizing across gels.
Fluorescent images are then overlaid to identify differentially ex-
pressed proteins. Software such as DeCyderTM software (GE Health-
care) uses the information gathered from the Cy2 labeled protein
standard across gels to determine which proteins are consistently
regulated across multiple gels. An advantage of gel-based ap-
proaches is that information on post-translational modications
(PMTs) can be readily extracted. Disadvantages of the gel based
methods include limited ability to quantify proteins that (1) are
highly acidic/basic, (2) have low/high molecular weight, (3) are
membrane bound proteins that are not soluble and readily frac-
tionated, (4) co-migrate with other proteins and (5) are not abun-
dant, although this last point remains a challenge for gel-free
methods as well.
3. Non gel-based, labeling approaches for protein quantitation
3.1. Isotope labeling of proteins with SILAC and ICAT methods
Both these methods utilize a light and heavy isotope to label
different protein mixtures. Stable isotope labeling with amino
acids in cell culture (SILAC) incorporates a light or heavy labeled
amino acid into the proteome of two different cell populations
growing in cell culturing media (e.g., 12C- and 13C-labeled L-lysine
or arginine) (Ong et al., 2002). As cells grow and divide, the labeled
amino acid is incorporated into proteins without compromising
protein function. The cell cultures can then be used to perform pro-
teomic proling for example, in prostate cell lines during cancer
progression (Everley et al., 2004). Krijgsveld et al. (2003) has ap-
plied this principle to labeling the proteome of higher organisms,
rst labeling Escherichia coli in medium enriched with 15N and
using the labeled E. coli as a food source for C. elegans. An analysis
of the proteome using 2D gel electrophoresis and MALDI-TOF
showed 95% incorporation of 15N in the rst generation of C. ele-
gans, increasing to 98% in the second generation. The authors also
successfully labeled the proteome of D. melanogaster using this
method.
Isotope coded afnity tags (ICAT) (ICAT reagents; Applied Bio-
systems, Inc., Foster City, CA) was also developed on the principle
that differences in isotopically labeled peptides could be resolved
and compared between two groups (Gygi et al., 1999a). Free cys-
teine thiol groups in peptides are targeted by the iodoacetamide-
based ICAT reagent under strong reducing conditions with either
the heavy or light isotope tag. An advantage of the ICAT reagents
is that, after digestion and mixing of the labeled peptides, one
can further separate the mixture through a streptavidin column
because each ICAT label has a biotin covalently linked to an ethyl-
ene glycol linker group. This feature further reduces the complex-
ity of the protein mixture before peptide identication and
removes non-labeled peptides from the experimental workow.
ICAT labeled peptide pairs are then distinguished by MS/MS. A lim-
itation of ICAT is that the peptide must have a cysteine present for
labeling to occur. Therefore, ICAT may label only relatively few
peptides of a protein, leading to fewer peptides available for
quantitation.
3.2. Isobaric tagging for relative and absolute quantitation (iTRAQ)
More recently, Applied Biosystems has developed a strategy
called isobaric tagging for relative and absolute quantitation of
proteins (iTRAQ) (Ross et al., 2004). The functional components
of the isobaric tagging system includes a reporter tag with varying
mass, a balance to insure labeled peptides from different treat-
ments have identical mass, and a peptide reactive group that
chemically tags amine groups of peptides generated from tryptic
digests. The 4-plex contains reporter tags 114117 m/z and more
recently, an 8-plex system has been developed with reporter tags
113119 and 121 m/z to quantify protein expression. Fragmenta-
tion of the peptide tag generates the low molecular mass reporter
ion and measurement of the intensity of these reporter ions en-
ables relative quantitation across treatment groups using algo-
rithms within programs such as Protein Pilot (Paragon TM TM
algorithm, Applied Biosystems). Advantages of this method include
increased ability to multiplex in a single LC MS/MS experiment and
increased coverage of the proteome because all peptides are theo-
retically labeled in contrast to ICAT which requires the presence of
cysteine groups. In addition, multiple peptides can be quantied
for a single protein and condence intervals can be calculated to
obtain statistical measures of protein changes.
Our group has recently used the iTRAQ method successfully in
a non-model teleost, the fathead minnow (FHM) (Pimephales prom-
elas), to investigate changes in protein abundance in the liver (Mar-
tyniuk, unpublished data). The purpose of the experiment was to
investigate the effects of an androgenic chemical, 17b-trenbolone,
on proteins in the liver. Female FHM were exposed to a dose of
5.0 lg/L 17b-trenbolone for a 48 h period in a ow through expo-
sure system. 17b-trenbolone is a potent AR agonist that binds
the AR with higher afnity than testosterone in FHM (Ankley
et al., 2003). Proteins were isolated using RIPA buffer (Pierce, Ther-
mo Fisher Scientic Inc., Rockford, IL, USA) and precipitated with
six volumes of acetone from six individuals; three control and
three 17b-trenbolone treated animals. Three separate iTRAQ
labeling reactions (randomly pairing a control and treated sample
 C.J. Martyniuk, N.D. Denslow / General and Comparative Endocrinology 164 (2009) 135141
for each reaction) were processed individually according to the
manufacturers protocol (Applied Biosystems). The process in-
cludes trypsin digestion of the proteins prior to labeling. The label-
ing procedure does not tolerate detergents and must follow the
prescribed method set up for optimal labeling by Applied Biosys-
tems. For each reaction, the control sample was labeled with tag
114 and the treated sample was labeled with tag 115. The sample
was desalted on a Vydac Silica C18 column (The Nest Group Inc.,
Southboro, MA) and the eluted peptides were dried and resus-
pended in 100 ll buffer A (75% 0.01 M ammonium formate and
25% acetonitrile) for off-line SCX fractionation using a polysulfo-
ethylA column (100  2.1 mm, 5 lm, 300 ). Peptides were eluted
using a linear gradient of 020% buffer B (75% 0.5 M ammonium
formate, 25% ACN) over 40 min, followed by a gradient of 20
100% buffer B for 5 min. Twenty fractions were collected and
pooled into four groups of about equal protein content for each
of the iTRAQ labeling experiments. Each of the 12 pools (four pools
from each of three iTRAQ reactions) was injected onto a capillary
trap LC Packings PepMap (DIONEX, Sunnyvale, CA) and desalted
for 5 min with a ow rate of 20 lL/min of 3% ACN, 0.1% acetic acid,
0.01% TFA and loaded onto the in-line LC Packing C18 Pep Map
HPLC column (300 lM  5 mm) connected to a QSTAR XL mass
spectrometer (Applied Biosystems). The peptides were eluted with
a ow rate of 200 nl/min with a 2 h elution gradient starting at 3%
solvent B and nishing at 60% solvent B (0.1% acetic acid, 96.9%
ACN).
The focusing potential and ion spray voltage on the mass spec-
trometer were set to 275 and 2600 V, respectively. The informa-
tion-dependent acquisition mode of operation was employed in
which a survey scan from m/z 4001200 was acquired followed
by collision induced dissociation (CID) of the three most intense
ions. Survey and MS/MS spectra for each IDA cycle were accumu-
lated for 1 and 3 s, respectively.
We detected peptides from approximately 293 liver proteins of
which 15 proteins were differentially altered after 17b-trenbolone
treatment. Proteins were identied using a homology based-strat-
egy from a tryptic peptide database constructed by us for all ray-
nned sh protein sequences available at NCBI. We used Mascot
(v2.2, Matrix Science, London, UK) and Protein PilotTM (ParagonTM
algorithm, v2.0, Applied Biosystem Inc.) software for this purpose.
Only proteins containing a least three high quality MS/MS peptides
were used in relative quantitation. A false discovery rate for pep-
tideprotein assignments was determined using both a reverse
dataset created by Proteomics System Performance Evaluation
Pipeline (ProteomicS PEP) in Protein PilotTM and a decoy database
as outlined by Elias and Gygi (2007). A FDR = 5% was considered a
positive peptideprotein assignment.
The number of total FHM liver proteins identied and quanti-
ed by LC MS/MS are comparable to iTRAQ studies done in rat
(Fig. 2). For example, in a study by Glckmann et al. (2007) 10
wk old Winstar male rats were administered N-nitrosomorpholine
to induce liver carcinogenicity and they identied 685 proteins. In
that study, the iTRAQ labeled peptides were separated only in one
dimension using a C18 column (PepMapTM) on an UltiMateTM nano-
LC system (Dionex). They then identied the proteins using a MAL-
DI TOF/TOF mass spectrometer (Applied Biosystems 4700/4800
Proteomics Analyzer). Of the identied proteins, 98 were deter-
mined to be differentially expressed, but 65% of their identied
proteins had only one peptide identied, 7% had 510 peptides
identied, and 6% of the proteins had greater than 10 peptides as-
signed to a protein. In our study with FHM liver, 293 proteins were
identied, of which 28% had one peptide assigned, 20% had 510
peptides assigned, and 14% had >10 peptides identied per protein.
This illustrates the importance of multiple dimensional separations
as this increases the overall number of identied peptides that can
be used to quantify differential expression. Increased protein iden-
137
Fig. 2. Comparison of the number of peptides assigned to a protein in an iTRAQ
experiment in rat liver (Glckmann et al., 2007) and fathead minnow liver
(Martyniuk, unpublished data). The difference in the number of peptides assigned
to a protein in the FHM liver compared to the rat is likely due to the larger number
of fractions analyzed in the FHM study and the separation approaches used in each
study.
tication and quantitation is likely to improve with database
searches specically to FHM (or the sh model of interest) as more
sh genomes are completed. Applications of iTRAQ have been
successful in other non-model vertebrates, for example Rana cates-
beiana to identify protein changes during metamorphosis and tail
reabsorption (Domanski and Helbing, 2007). This study and ours
demonstrate that this quantitative approach can be used in non-
model organisms in which genomic data is limited.
To further explore the cellular processes altered by 17b-trenbo-
lone in the FHM liver, pathway analysis was done using Pathway
Studio (v5.0) (Nikitin et al., 2003; Ariadne Genomics) (Fig. 3). This
approach provides novel information on proteinprotein interac-
tions, common regulators of differentially modulated proteins,
and common targets based on literature searches. Pathways are
built by nding the shortest paths between selected entities using
databases in Pathway Studio. Cellular pathways altered by 17b-
trenbolone include glycolysis and cell division and were involved
in processes such as liver dysfunction and cancer. These data sug-
gest that these pathways are regulated in part by AR signaling in
the FHM liver. Other programs for pathway analysis include KEGG
(Kyoto Encyclopedia of Genes and Genomes) and Ingenuity Path-
ways Analysis (Ingenuity Systems, Redwood City, CA, USA) and
are useful tools in placing molecular data into a physiological con-
text. Bioinformatics approaches are critical for integrating genomic
and proteomic data and provide additional insight into functional
changes that can occur after a treatment, such as androgenic
chemicals.
4. Non gel-based, label-free approaches for protein quantitation
4.1. Normalized ion current and peptide ion intensities
Label-free approaches for protein quantitation also depend on
successful reduction of the proteome using multiple fractionation
techniques, such as MudPit. Once fractionation is preformed, one
is able to utilize the peptide ion intensity to quantify relative pep-
tide abundance because the intensity of m/z is related to the abun-
dance of the peptide. Thus, peak ion intensities extracted from
mass spectrum provide a relative measure of protein abundance
(Cutillas and Vanhaesebroeck, 2007). These authors improve the
condence of their quantitation by determining the mean peptide
ion intensity for a number of peptides from a given protein, since
138 C.J. Martyniuk, N.D. Denslow / General and Comparative Endocrinology 164 (2009) 135141

Fig. 3. Pathway studio analyzes of proteins regulated by the androgenic compound 17b-trenbolone in FHM liver. Also included in the gure are the relationships between
proteins and the androgen receptor. The purple line represents direct protein binding, the blue line represents a relationship due to expression, and the gray line represents a
relationship due to regulation. Cellular pathways altered by 17b-trenbolone include glycolysis and cell division along with proteins implicated in liver dysfunction and cancer.
Abbreviations are as follows. AGMAT, agmatine ureohydrolase; AR, androgen receptor isoform 1; CALM3, calmodulin; COL1A1, collagen a1; FABP6, liver fatty acid binding
protein; FAH, fumarylacetoacetate hydrolase (fumarylacetoacetase); GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBA2, hemoglobin alpha chain; HSPD1, heat shock
60 kD protein 1; SOD1, Cu/Zn superoxide dismutase; TPI1, triosephosphate isomerase 1b.

individual peptides ionize with different efciencies. An important
consideration is that ion intensities must rst be normalized to an
internal standard. This method has also been applied to identify
phosphorylation sites (Steen et al., 2005).
4.2. Spectral counting and absolute quantitation (AQUA)
Spectral counting is a semi-quantitative method that exploits
the correlation between the number of observed peptides identi-
ed for each protein by LC MS/MS and the abundance of the pro-
tein (Gao et al., 2005). Also known as the peptide hit technique;
spectral counting requires some calibration and adjustment due
to the length of each protein to adjust the spectral counts. In gen-
eral, the ratio of a protein is equal to [(nB + 1)(tA/tB)/(nA + 1)]
where nB and nA are the total number of spectra identied for a
particular protein in sample B and A, respectively. The normaliza-
tion factor tA/tB utilizes the total number of identied spectra for
all proteins in A or B and acts as a weighted adjustment. This tech-
nique is optimal with genomes that are completely sequenced so in
practice, all peptide spectra can be assigned to a particular protein.
This avoids bias from spectra that are detected but cannot be as-
signed. This method may not be ideal for non-model species where
limited genomic information is available.
Absolute quantitation (AQUA) is also a label-free method that
utilizes a heavy labeled peptide standard mixed with the tryptic
protein digest of the sample for quantitation (Gerber et al.,
2003). A standard probe or spike peptide of a specic tryptic pep-
tide from the protein of interest is synthesized with one amino acid
containing a heavy isotope. The spike peptide must be ionized and
detected consistently in a complex protein mixture and must be
unique to the protein of interest. A standard curve is then gener-
ated with known amounts of the heavy peptide. Standard AQUA
curves can be extremely sensitive and accurate, down to the fento-
mol range with a high R2 = 0.998 (Ottens et al., 2007). To quantify a
protein, a known amount of the reference or spike is added to a
complex protein mixture and the ion intensity of the spike (heavy
isotope) is compared to the ion intensity of the unknown sample
(light isotope). A standard curve bracketing the concentrations of
the unknown sample is constructed for precise quantitation. Theo-
retically, one could generate multiple AQUA probes to spike a sam-
ple and quantify many proteins within the same mixture.
However, a limitation to this method is the multiple individual
standard curves that must be generated for each protein to be
quantied. Another limitation is that often low abundant proteins
maybe undetectable in a complex mixture. This problem can cir-
cumvented by enriching for the protein of interest (Warren et al.,
2005).
5. Comparison of quantitative proteomic techniques
An important question to address is how different proteomic
methods compare to each other in the number of proteins identi-
 C.J. Martyniuk, N.D. Denslow / General and Comparative Endocrinology 164 (2009) 135141
ed and quantied. Glckmann et al. (2007) compared data from
both 2D gel electrophoresis (2DE/MS) and iTRAQ methods after
rats were treated with the carcinogen N-nitrosomorpholine. Using
2DE/MS, 57 proteins were identied as differentially expressed
while the iTRAQ method identied 98 proteins. Of the biomarkers
identied, 63% (36) of the proteins were identied by both tech-
niques and the expression of 26 proteins were conrmed by both
2DE/MS and iTRAQ. This suggests that both methods work well
and are complementary in the number of proteins identied.
Using both ICAT and iTRAQ labeling, DeSouza et al. (2005)
identied common protein markers in uterine tissue with endome-
trial carcinoma, and showed that the proteomic information gener-
ated by the two methods was largely complementary and
identied similar number of housekeeping and metabolic proteins.
However, there were some differences in the functional classes of
proteins identied between the two methods. iTRAQ identied
approximately twice the number of proteins involved in transcrip-
tion/translation while ICAT identied approximately twice the
number of proteins involved in cell signaling, when compared to
each other. This was most likely because the iTRAQ method iden-
ties a larger percentage of the ribosomal proteins than ICAT.
Schmidt et al. (2004) compared ICAT-LC/MS with 2DE/MS to
identify proteins in M. tuberculosis. The authors found that each
method was biased towards a particular functional class of pro-
teins. The ICAT method preferentially identied proteins involved
in intermediary metabolism, non-ribosomal peptide synthesis
and transport/binding proteins while the 2DE/MS method identi-
ed more proteins involved in lipid biosynthesis, chaperones/
heat-shock proteins, and protein secretion. In the same study,
2DE/MS identied 108 proteins and ICAT identied 280 proteins
with 27 proteins identied using both techniques. Thus, although
some methods such as iTRAQ provide increased protein identi-
cation and greater range in expression changes compared to 2DE/
MS (Choe et al., 2005), each method provides additional informa-
tion that increases overall understanding of the underlying biology.
6. How does the proteome relate to the transcriptome?
One important aspect of functional genomics is to determine
how gene expression is correlated to protein translation. Currently,
it is poorly understood how well an increase in mRNA steady state
abundance correlates with increased protein abundance. Each
mRNA and protein has intrinsic steady state levels that depend
on both synthesis and degradation and these half lives may not
be comparable. Some proteins may be more long lived than their
corresponding mRNAs and these proteins may be informative bio-
markers. This is active area of research in environmental toxicology
and studies are beginning to consider changes at both the genome
and proteome in sh toxicological studies (Martyniuk, unpublished
data; De Wit et al., 2008). Biomarkers for contaminant exposures
may be resolved or improved by considering both transcriptomic
and proteomic changes.
Pearson and Spearman-Rank correlations have been used to
measure the response of the transcriptome in relation to the prote-
ome. Hack (2004) did a survey of published literature that com-
pared genomic and proteomic data sets using Pearson and
Spearman-Rank correlations. Both Serial Analysis of Gene Expres-
sion (SAGE) and microarray data in general showed between 45
75% similarity when compared to quantitative proteomic data.
Gygi et al. (1999b) investigated the correlation between protein
and mRNA levels for 106 genes in yeast growing in log phase with
glucose as a carbon source. The Pearson product moment correla-
tion coefcient was r = 0.935 for all 106 genes and proteins. How-
ever, this estimate was biased by small numbers of genes/proteins
with high abundance. When considering only the lowest expressed
139
4095 proteins, the correlation values ranged between r = 0.10.4.
In another study, Minagawa et al. (2008) compared the genomic
and proteomic responses in human patients that had hepatocellu-
lar carcinoma (HCC). The authors utilized a 2D DIGE approach and
identied 125 proteins using an electrospray ionization ion-trap
mass spectrometer (LCQ Deca XP). The average HCC/non-HCC
expression ratios of 93 proteins were plotted against mRNA ratios
and the Pearsons correlation between the two was 0.73. Further
insight was gained by separating proteins rst by function and
comparing this to gene expression. There was a high correlation
(r = 0.90) for transcripts and proteins involved in cell metabolism
while the remaining proteins showed less of a relationship to tran-
script levels (r = 0.36). Thus, it appears as though there is some cor-
relation between the steady state mRNA abundance and protein
abundance. However, the analytical methods used, the categorical
function of genes/proteins, complex transcriptional/translational
regulation, post-translational modication of proteins and tempo-
ral effects inuence the relationship between genome and
proteome.
7. OMICS and reproductive steroid receptor regulation in sh
Microarray based studies have provided a signicant amount of
information on the molecular events underlying steroid signaling
in sh. This includes studies using E2, pharmaceutical estrogens
and androgens (e.g., 17a-ethinylestradiol, 17a-methyldihydrotes-
tosterone, dihydrotestosterone), or chemicals that are steroid
mimics (bisphenol A, nonyphenol). Genomic approaches for study-
ing reproductive steroid effects in sh are growing and have been
investigated in tissues that include the hypothalamus (Martyniuk
et al., 2006; Marlatt et al., 2008), telencephalon (Martyniuk et al.,
2007), liver (Moens et al., 2007; Benninghoff and Williams, 2008;
Hoffmann et al., 2006, 2008), and gonad (Santos et al., 2007). Tran-
scriptomic studies suggest that sh tissues have unique genomic
responses to estrogens (Martyniuk et al., 2007; Garcia-Reyero
et al., 2008). However, there are common cellular processes af-
fected in multiple tissues that can be regulated, for example lipid
metabolism and transport, cellular respiration and metabolism,
electron transport, DNA/protein binding, and protein synthesis by
estrogens.
Despite the signicant genomic advances, quantitative proteo-
mic-based studies in sh are few and have been limited to issues
in aquatic toxicology. 2DE/MS analysis identied 15 proteins in
the rainbow trout liver proteome that were altered after sewage
treatment water efuent (Albertsson et al., 2007). In another study,
E2 and 4-nonylphenol exposures of embryonic zebrash had un-
ique proteomic ngerprints as analyzed by 2DE/MS but the pro-
teins were not identied (Shrader et al., 2003). More recently
developed techniques such as DIGE and ICAT have been applied
successfully to study the proteomic response to hypoxia in medaka
(Oryzias latipes) (Oehlers et al., 2007) and disease infection in
Atlantic salmon (Salmo salar) (Booy et al., 2005) respectively. How-
ever, the effects of reproductive steroids and environmental mim-
ics on the sh proteome remain largely an open area of research.
8. Summary
Fish physiology and toxicology are moving towards the integra-
tion of genomic, proteomic, and metabolomic data sets to better
understand the underlying physiology and how animals interact
with their environment. In sh, studies are beginning to character-
ize the proteome of tissues such as liver for further study (Wang
et al., 2007). Bioinformatics approaches such as pathway analysis
will continue to be important in providing functional insight into
genomics and proteomics.
140 C.J. Martyniuk, N.D. Denslow / General and Comparative Endocrinology 164 (2009) 135141
The application of proteomics to the response of sh tissues to
steroids must consider that the various proteomic techniques are
complementary but will yield different information due to separa-
tion and fractionation protocols, label-free or label methods, MS/
MS approach, and database construction and search engines.
Although there is the capability to study proteomic responses in
sh, many non-gel based approaches in quantitative proteomics
offer limited information on post-translational modications
which is essential for protein function, for example protein meth-
ylation or phosphorylation.
Acknowledgments
We thank collaborators S. Alvarez and S. McClung at the Inter-
disciplinary Center for Biotechnology Research at the University of
Florida for assistance in developing the iTRAQ method and adapt-
ing it for use to a teleost model. We also thank collaborators
G. Ankley and D. Villeneuve at the Environmental Protection
Agency in Duluth, MN for sh exposures and VL Trudeau at the
University of Ottawa, co-chair for the 6th International Symposium
on Fish Endocrinology: Functional Genomics Symposium. This re-
view was supported by NSERC PDF to CJM and the Environmental
Protection Agency STAR grant (R831848) to ND.
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