|Molecular Vision 2006;
Received 8 February 2005 | Accepted 25 April 2006 | Published 12 June 2006
Proteomic analysis of opsins and thyroid hormone-induced retinal development using isotope-coded affinity tags (ICAT) and mass spectrometry
W. Ted Allison, Kathy
M. Veldhoen, Craig W. Hawryshyn
Department of Biology, University of Victoria, Victoria, British Columbia, Canada
Correspondence to: Dr. Craig W. Hawryshyn, Department of Biology, Queen's
University, Kingston, Ontario, Canada, K7L 3N6; Phone: (613) 533-6000
x78529; FAX: (613) 533-6617; email: email@example.com.
Dr. Allison is now at the Department of Molecular, Cellular & Developmental Biology, Ann Arbor, MI.
Purpose: Analyses that reveal the relative abundance of proteins are informative in elucidating mechanisms of retinal development and disease progression. However, popular high-throughput proteomic methods do not reliably detect opsin protein abundance, which serve as markers of photoreceptor differentiation. We utilized thyroid-hormone (TH) treatment of rainbow trout (Oncorhynchus mykiss) as a model of cone apoptosis and cone regeneration. We used this model to investigate if emerging proteomic technology allows effective analysis of retinal development and opsin protein abundance. We also sought to begin a characterization of proteomic changes in the retina occurring with TH treatment and address whether TH affects proliferation or photoreceptor differentiation.
Methods: Retinal homogenates were prepared from control and TH-treated fish. Peptides from control and treated homogenates were differentially labeled, using isotope-code affinity tags (ICAT) and analyzed using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry (capLC-ESI-MS/MS). This method identifies proteins and quantifies their relative abundance between two samples.
Results: The relative abundance of many retinal proteins changed during TH treatment. These included proteins from every functional class. We detected 1,684 different peptides, and our quantification suggests that 94 increased and 146 decreased in abundance more than 50% during TH treatment. Cell-cycle proteins appear to be increased, consistent with TH-inducing cell proliferation, similar to its effect in Xenopus. Other proteins associated with retinal development, such as ΔA and tubulins, changed in abundance during TH treatment. Rod opsin and three cone opsins were identified and the relative abundance of each changed with TH treatment.
Conclusions: ICAT and capLC-ESI-MS/MS are an effective complement to other molecular approaches that investigate the mechanisms of retinal development. Unlike other proteomic techniques, this approach does not require development of species- or tissue-specific methodology, such as characterizing two dimensional (2D) gels or antibodies, in order to be practical as a high-throughput approach. Importantly, this technology was able to assess the relative abundance of opsin proteins. These findings represent the first high-throughput proteomic analysis of the retina and demonstrate the technique's ability to provide useful information in retinal development.
The characterization of retinal proteins is valuable in understanding mechanisms of retinal development, disease and repair. Such studies can be effective complements or alternatives to high-throughput analyses of the transcriptome such as those accomplished by microarrays and serial analysis of gene expression [1-3]. For example, proteomic analyses may be favoured over transcriptome analyses when mRNA abundance does not reflect protein abundance . Furthermore, transcriptome analysis is unable to assess post-translational modifications that can alter protein function.
To date, characterization of the retinal proteome has been accomplished using two dimensional (2D) gels or panels of antibodies. Recent efforts on several animal models have focused on measures that will allow these strategies to become high-throughput. One approach has been to catalog the identities of protein spots on 2-D gels. This approach has established the prerequisite background to allow characterization of porcine, bovine, and murine retinal proteomes [1,5-8]. Other efforts have focussed on characterizing panels of relevant antibodies for their utility in zebrafish immunohistochemistry ; at least for some targets, this approach can be quantitative .
We utilized Oncorhynchus mykiss, a salmonid fish, as a model of cone death and cone regeneration. During natural development, ultraviolet-sensitive (UVS) cones, which are homologs of mammalian "s-cones" or "blue cones," die from the retina of salmonids by apoptosis [11-14]. In Oncorhynchus mykiss, this UVS cone death appears to be associated with smoltification, a metamorphic transition that prepares migratory populations for physiological requirements of the marine environment. Thyroid hormone (TH) peaks during smoltification, and treatment of salmonids with TH can mimic many aspects of smoltification [15,16], including changes in the visual system such as UVS cone death [12,14,17-19]. Of notable significance, UVS cones reappear into the retina later in the salmonid life history, possibly during sexual maturation and the return migration to freshwater spawning grounds [12,14,18,20-22]. The quickly growing trout retina has proliferating cells in the established retina  that respond to injury [12,24,25] and support the regeneration of the UVS cone [12,14].
Both UVS cone death and regeneration can be induced by manipulations of TH [12,14,17-20,26]. This provides a controlled onset time for developmental events of interest. TH treatment is known to produce several other effects in the retina, including changes in rhodopsin/porphyropsin levels [27-30], cell size/density , proliferation [12,14,31,32], and changes in deiodinase levels . Deiodinases are enzymes that activate and deactivate circulating TH in an organ-specific manner. The retina of teleosts, Xenopus, and mammals express deiodinases [3,32-35] as measured by enzyme activity and in situ hybridization. Salmonid fishes, including rainbow trout, have surprisingly high levels of deiodinase activity , indicating that TH metabolism has a substantive role in salmonid retinal physiology and acts primarily at the level of retinal cells to effect initiate their development.
Analyses of retinal development in salmonids have mostly been limited to histology [11,17,18,20,21,36-46], electrophysiology [13,14,19,37,45,47-50], and transcriptome analyses [13,51]. We have recently worked with existing antibodies [12,14,25,52] and developed novel antibodies [12,14,53] to examine proteins in trout retinal development. Few other antibodies have been characterized in salmonid retina [54-65]. We sought a proteomic approach to investigate the unique developmental events in rainbow trout retina during TH treatment. Whereas several papers have been successful using 2D gels on other trout tissues [66-68], we noted that strategies using antibodies or 2D gels require substantial investment in preparatory experiments before high-throughput analyses can be fruitful. Antibodies do not allow discovery of novel gene products, a particular interest of ours in exploring our model of how stem cells regenerate cones . Further, we noted that the detection limits of 2-D gels prohibit reliable detection of opsin proteins. Indeed, several thorough analyses of retina using 2-D gels have failed to identify rod or cone opsins, despite rod opsin being one of the most abundant retinal proteins [1,5-8]. The ability to identify opsin abundance has often been used as an effective marker for rod and cone differentiation, and as a proxy for disease progression.
We have investigated an evolving proteomic technique that utilizes isotope-coded affinity tags (ICAT) and mass spectrometry (MS) to simultaneously quantify and identify peptides. The method reports relative abundances of peptides from two protein pools . All cysteine-containing proteins are labeled with a biotin-based tag (Figure 1). The tag has either a heavy or light isotope, owing to the presence of nine deuterium atoms in the heavy isotope, and thus the peptides from each pool differ in mass by 9 Da (Figure 1, Figure 2). The protein pools are combined and then trypsinized. Trypsin-derived peptides are examined in a tandem MS. Pairs of peptides from each protein pool are then recognized by their 9 Da mass differential, and their relative abundance can be quantified. The associated proteins are finally identified using collision-induced decomposition fragments of the selected peptides in interrogation of peptide fragment databases. The technique has only recently been applied to tissues as morphologically complex as the retina. Although this technique is known to be a quantitative methodology, with examples of this in the literature [69-74], our aim was to evaluate the methodology, look for differences, and present them for reference and as guideposts for future research. This research represents a starting point in the literature for future research.
We find that despite the relatively limited genetic databases currently available for our model organism, the technique revealed several peptides from proteins of unknown function and demonstrated that many proteins changed in abundance during TH treatment. Furthermore, the technique identified changes in the relative abundance of rod opsin and three cone opsin proteins.
Animals and sample preparation
Rainbow trout were obtained from the Vancouver Island Trout Hatchery, Duncan, British Columbia, Canada. Fish were housed at the University of Victoria Aquatics Facility and maintained at 15±1 °C. A 12 h light:12 h dark photoperiod was provided by fluorescent lights (Stanpro FU32T8/65K/8 6500K, Saint-Laurent, QC). Fish were maintained in these conditions for at least four weeks prior to sampling. Fish were fed three times per week with Moore-Clark Trout AB feed. Care of the fish and all procedures were in accordance with and approved by the University of Victoria Animal Care Committee under the auspices of the Canadian Council for Animal Care.
Rainbow trout parr were treated for nine days in an exogenous bath of L-thyroxine sodium salt (Sigma, St. Louis, MO) dissolved in 0.1 N NaOH to a final concentration of 300 μg/l. Water was changed daily. Control fish were treated identically, but only vehicle (NaOH without L-thyroxine) was added to their water. Control and TH-treated fish had average standard lengths (±1 standard deviation [SD]) of 79±9 and 82±9 mm (n=5), respectively.
Starting 4 h after initiation of the light cycle, trout were dark-adapted for 1 h, sacrificed by MS-222 overdose followed by cervical transection, and the neural retina was dissected in ice-cold PBS. Following dissection, the entire retinas were immediately placed in 500 μl of ice-cold buffer (30 mM Tris-HCl pH 7.5, 10 mM EGTA, 5 mM EDTA, 250 mM sucrose, 1% octylglucopyranoside [Sigma]). Each retina was then homogenized on ice, using a disposable Kontes Pellet Pestle with cordless motor tissue grinder (Kimble Kontes, Vineland, NJ) for approximately 1 min, vortexed for 15 s, and microcentrifuged at 4 °C at 10,000x g for 10 min. The resulting supernatant was collected and used for ICAT labeling.
Production and analysis of ICAT-labeled peptides
Production and analysis of ICAT-labeled peptides was performed by the University of Victoria Genome British Columbia Proteomics Center. All procedures were performed according to the manufacturer's recommended protocols using kits supplied by Applied Biosystems Inc., Foster City, CA. Control and treated retinal homogenates were solubilized in 0.1% SDS, 6 M urea, and protein quantification was performed using Bio-Rad's Protein Assay Dye Reagent Concentrate (Catalog number 500-3006). ICAT-labeled samples were then prepared using a Cleavable ICAT Reagent (Applied Biosystems, Bulk Kit Product No. 4337339), which specifically reacts with cysteine sulphydryls leaving an attached biotin group. Briefly, to equal amounts of control and TH-treated protein samples (100 μg), 80 μl of 25 mM ammonium bicarbonate buffer, pH 8.5 and 2 μl of 50 mM TCEP (Tris[2-carboxyethyl]phosphine hydrochloride) were added to enable full disulfide reduction. This reaction was allowed to proceed for 15 min at 37 °C, followed by addition of the appropriate ICAT reagent in 20 μl of acetonitrile. The latter reagent was allowed to react for another 2 h at 37 °C in the dark, whereupon light and heavy labeled samples were combined prior to digestion with 20 μg of chemically modified porcine trypsin (Promega, Madison, WI). Proteolysis occurred for 18 h at 37 °C, in 600 μl of 25 mM ammonium bicarbonate buffer, pH 8.5. Excess reagents were removed using an ICATTM Cation Exchange Buffer Pack and Cation Exchange Cartridge (Applied Biosystems) and biotin-labeled ICAT peptides were isolated by avidin affinity column chromatography using an Affinity Buffer Pack (Applied Biosystems). The biotin group was finally cleaved from eluted peptides using 95% TFA containing 5 μl of a proprietary scavenger. Reaction was allowed to proceed for 2 h at 37 °C. Samples were stored as lyophilized powders.
ICAT two dimensional liquid chromatography tandem mass spectrometry
Liquid chromatography systems (UltiMate gradient pumps, SwitchOS II and FAMOS Auto-sampler; LC Packings/Dionex, Amsterdam, The Netherlands) were controlled by Applied Biosystems Analyst software during the data collection. The ICAT-labeled samples were brought up in 40 μl of 5% acetonitrile/water containing 0.1% formic acid and loaded into auto-sampler vials. A 10 μl volume was loaded onto a 100 μl sample loop with the remainder of the volume being filled with 0.1% formic acid. The SwitchOS II loading pumps were set to a flow rate of 30 μl/min, and the sample was pumped onto a 500 μm x 15 mm BioX-SCX 5 μm strong cation exchange column connected, in turn, to a 300 μm x 1 mm PepMap C18, 100 Å nanoprecolumn (LC Packings/Dionex). The eluant from these columns was allowed to divert to waste for 7 min using the SwitchOS II. The SCX column was switched off-line and the sample was washed for another 7 min on the abovenamed PepMapTM C18 column to concentrate and desalt the peptide mixture before MS analysis. The eluant was then diverted to the UltiMate pumps, and the sample was eluted onto a 75 μm inner diameter (I.D.) x 15 cm PepMapTM 3 μm, 100 Å C18 nanocolumn (LC Packings/Dionex). The column was sleeved via 20 cm of 20 μm I.D. fused silica (Poly Micro Technologies, Phoenix, AZ) to a Valco stainless steel zero dead volume fitting which had the high voltage lead (2500 V) and a New Objective (Woburn, MA) emitter fused silica tip positioned at the orifice of a PE SCIEX API QSTAR Pulsar in positive ion mode (PE SCIEX, Concord, Ontario, Canada). The UltiMate pumps were set to deliver a flow rate of 150 nl/min with the following buffers: solvent A: 0.1% formic acid/water; solvent B: 80% acetonitrile/20% water/0.1% formic acid. The gradient used to elute the peptides was 15 min at 0% B, 30 min to 60% B, 3 min to 80% B and held for 2 min, 3 min to 0% B, and 8 min to re-equilibrate the column. The SCX column was then switched back in-line with the SwitchOS II buffer of 0.1% formic acid/water to equilibrate for the next injection.
To elute further peptides from the SCX column, 50 μl volumes of 0.5% formic containing from 100 to 1000 mM ammonium acetate, pH 4.0, was applied successively in 100 μm increments, finishing with a 2 M solution. The above described organic modifier gradient was then applied after each salt injection.
The mass spectrometer information-dependent acquisition parameters were as follows: After a 1 s survey scan from 300-1500 m/z peaks with signal intensity over 10 counts with charge state 2-5 were selected for MS/MS fragmentation using a software determined collision energy and then a 2 s MS/MS from 65-1800 m/z was collected for the two most intense ions in the survey scan. Once an ion was selected for MS/MS fragmentation it was put on an exclude list for 180 s to prevent that ion from being gated again. A 6 amu peak window was used to prevent gating of masses from the same isotopic cluster during the survey scan.
The software used to analyze the data was ProICAT SP2 (Applied Biosystems software is version 1.1). The National Center for Biotechnology Information (NCBI) non-redundant database was searched with an error tolerance of 0.15 Da for both the MS and the MS/MS scans. Also searched were databases provided by the Genomic Research on Atlantic Salmon Project (GRASP). The results were then written by the software to a Microsoft Access database. The results database was then queried with a minimum confidence limit of 50 and a score of 15. The number of sequences in the databases at the time searched was 1,934,002 for NCBI, 13,352 for the Salmo salar GRASP, and 19,509 for the Oncorhynchus mykiss GRASP databases. The only fixed modification considered was the cysteine ICAT labels, MS and MS/MS tolerances were set to 0.15, and one missed cleavage was permitted. Nontryptic peptides were not considered.
Experimental and control proteins were labeled independently and subsequently mixed together. Therefore, both heavy and light protein adducts are digested under exactly the same conditions and resultant peptides measured relative to one another. Under these circumstances, any variation in proteolytic digestion would be observed in both heavy and light forms and thus be irrelevant. Moreover, control and experimental peptides were separated under identical LC and MS separation conditions. As such, relative measurements were reliably obtained and the major source of error can be attributed to biological variation of the proteome.
The Interrogator peptide database search algorithm of the ProICAT 1.0 SP2 software (Applied Biosystems, Foster City, CA). For each database peptide, within tolerance of the observed parent mass, the algorithm rapidly counts the theoretically expected ions found in the spectrum. The score reported for each database peptide is the sum of intensity-based weights for all matching fragments. From the distribution of top scores for a particular spectrum, a score deemed representative of the best random peptide hits is found, and a distance score is computed as the difference between the top peptide score and this best random score. Discriminant analysis of score, distance score, and other metrics was performed on annotated data to determine a discriminant function that is effective at discriminating right answers from wrong. Identification confidence is reported based on the observed rate of correct identifications for various ranges of the discriminant function. Confidence is based upon both the score and distance score and is dependent upon both the number of matching ions and the distance of the matched peptide from other matches for the same MS/MS data. Protein expression ratios are calculated from the MS spectra using an LC/MS spot-finding technique to detect and quantify the abundances of peptides. ICAT reagent expression pairs and isolated peptides are found. A quality value for each quantification result is computed for complete pairs based on the accuracy of the measured mass difference and the similarity of light and heavy isotopic profiles. Finally, ProICAT associated MS-based peptide expression measurements with MS/MS-based protein identifications to compute expression ratios for each protein.
The ICAT method has been intensively studied by a number of labs. When equal amounts of heavy and light congeners are analyzed by ESI-MS or MALDI-TOF MS one invariably obtains ratios of 1:1, within pipetting and instrument error . Indeed this criterion of performance is monitored whenever a new stable isotope procedure is evaluated.
In our results, the protein accession number and name represent the raw software output. Other proteins that contain the exact peptide described, including those from other species, are equally likely to represent the actual protein that was detected in the trout retina. The peptides reported in the results section can be searched against protein database (e.g., NCBI's protein-protein BLAST) for alternate proteins that contain the peptide.
We demonstrate that ICAT combined with 2D LC-ESI-MS/MS is a rapid method to compare the relative abundance of proteins between two retinal samples. Consistent with previous reports [75-77], we find that the technique provides broad coverage of the proteome. In addition, previous reports detailing the performance of the University of Victoria Genome British Columbia Proteomics Center indicated a strong correlation between the relative abundance of proteins reported by ICAT combined with LC-ESI-MS/MS, and results of western blots or 2-D gels [74,76].
We found that fractionation of the sample led to greater coverage of the proteome, as compared to our preliminary experiments, where fractionation of the samples was not employed.
A D9:D0 ratio (Heavy ICAT isotope: Light ICAT isotope), differing by the inclusion of 9 deuterium ions in the heavy isotope, (Figure 1) greater than 1.0 represents peptides that were more abundant in the TH-treated retina, and ratios less than 1.0 are peptides that were more abundant in the control retina. Standards run with this ICAT protocol at the University of Victoria Genome British Columbia Proteomics Center indicate that it can reliably detect a 30% change in protein abundance. We therefore chose a criterion fold change of 50% to represent a change in relative protein abundance during TH treatment. Similar to a previous study using this proteomics facility , we chose this criterion to be a 50% change in relative abundance. Therefore, a peptide met our criterion if the D9:D0 ratio was greater than 1.5 or less than 0.667. In our results the ratio D9:D0 is equivalent to T:C ratio, where T represents the abundance of the peptide in thyroid hormone treated retina, and C represents the abundance in the control retina. Our criterion is arbitrary; some proteins that do not meet it may still be changing in a measurable way that is biologically meaningful. The data in Table 1 and Appendix 1 are intentionally presented in order of the fold change for each peptide. Thus, one can quickly see which peptides are above or below a particular criterion. This proteomic approach serves as an effective tool to explore changes in a proteome and the regulation of individual proteins can be validated using other techniques if they are of particular interest. A peptide was considered "quantified" if a ratio was reported by the software. Other peptides were detected in only one of the two pools, and therefore no ratio is available. Still other peptides were identified but not quantitated. This could be due to incomplete labeling, low signal-to-noise ratios, and/or incomplete elution during fractionation or purification stages.
We further categorized peptides as belonging to proteins that were either "characterized" or "uncharacterized." Uncharacterized proteins represent results where the protein is unnamed or listed as "hypothetical" or "putative." Further uncharacterized proteins included those submitted to databases based on large-scale nucleotide sequencing, such as those beginning with the prefix "ENSANGP" or those identified by the RIKEN mouse cDNA project. Alternatively, we describe proteins as characterized if they have been named and their function described/predicted.
We also included as "characterized" all proteins that were detected when the MS data were processed against the salmonid EST databases of GRASP. Some of these proteins are not named: however, we know that they are expressed in salmonids as mRNA that conceptually predict the exact peptides. For peptides found in the GRASP database, the name of the protein is followed by the unique GRASP identifier for the cDNA clone and the nucleotide accession number. The GRASP sequences comprise three salmonid species that can be discerned from their unique identifiers (ssal represents Salmo salar, Atlantic salmon; omyk represents Oncorhynchus mykiss, rainbow trout; oner represents Oncorhynchus nerka, sockeye salmon). For the genes from GRASP, the GenInfo Identifier (GI) number that is listed often represents a homolog of the gene from another species. For example, in Table 1, a peptide that is part of the protein cyclin I was increased 11 fold by TH treatment. The 12 amino acid peptide is an exact match to a cDNA from Atlantic salmon (Salmo salar). This cDNA has been identified, by sequence identity over its entire length, to be a homolog of the mouse cyclin I gene; thus, the mouse cyclin I gi number is listed. One may query the GRASP project for further details of individual cDNAs, including using the accession number we report to search their online database of gene sequences (GRASP) associated with their cDNA microarrays .
In this study, we detected 1,684 unique peptides, varying from 5 to 29 amino acids in length (Figure 3, Table 1, Table 2, Appendix 1). The results, when comparing TH-treated to control retina, suggest that 70 characterized proteins (Table 1), and a further 24 uncharacterized proteins (Table C in Appendix 1), increased by more than 1.5 fold. TH treatment led to a decrease in abundance of 122 characterized proteins (Table 1) and 24 uncharacterized proteins (Table C in Appendix 1).
We quantified many proteins that changed less than 50% in their relative abundance during TH treatment, including 321 characterized (Table A in Appendix 1) and 268 uncharacterized proteins (Table D in Appendix 1). The majority of peptides (i.e., 854) were not quantifiable or were detected in one sample only (Tables B and E in Appendix 1).
To assess the reliability of the technique, we examined multiple peptides that originated from the same protein to see if they changed in a similar manner. Nine proteins were detected more than twice (Table 3). These are reported along with the average and SD of the quantification for each peptide. Seven of these groups of peptides had minimal variation with standard deviations less than 0.16. Two others proteins have more variation among peptides, although the standard deviations remain small (<0.37) relative to our criterion (0.5).
Twelve proteins were detected twice, and are reported with the average and difference in the quantifications. The average of these differences (0.144) is small relative to our criterion (0.5). In one instance, the variation (0.786) is larger than our criterion. Until genomic sequencing is complete for rainbow trout we will be unable to eliminate the possibility that some of the peptides in Table 3 originate from different proteins. Thus, our data appear to be reliable en masse; however, each result should be interpreted with caution until it is confirmed with other methods.
We have summarized the TH-induced changes to opsin proteins in Table 4. Changes were observed in four of the five opsin classes known to occur in rainbow trout  and other salmonids . Four peptides from three cone opsins were unambiguously identified, as were two peptides from rod opsins. The expression of these opsins in particular photoreceptor classes has been accomplished using in situ hybridization  and immunohistochemistry . Our results suggest that LWS and MWS opsins were more abundant (1.9- and 2.5 fold, respectively) in TH-treated fish. Two peptides were detected from SWS2 opsin, one of which met our criterion for increased abundance (1.5 fold) and the other showed similar levels but was increased only 1.3 fold. Peptides were also detected from the rod opsin gene RH1.
Effects of thyroid hormone in the retina
TH is known to have several effects in the retina. In Xenopus, TH leads to increased proliferation in the retinal margins [31,32]. A similar observation has been made via analyzing BrdU incorporation in rainbow trout retina . TH treatment also leads to increased proliferation in other salmonid sensory epithelia, including coho salmon olfactory epithelium . Our results suggest that TH may promote proliferation in the retina. Most notably, cyclin I increased with TH treatment (Table 1). This was supported by another peptide, G-2, and S-phase expressed 1 (GTSE-1), which increased in abundance. GTSE-1 is expressed specifically during cell cycle phases S and G2 [81,82].
TH is also known to affect the density of photoreceptors in the trout retina . Our results indicated that peptides derived from various tubulin subunits changed in abundance during TH treatment. In particular, three peptides decreased in abundance. Ten peptides attributed to tubulins did not meet our criterion of 50% fold change and appear in Table 3 and Appendix 1. Microtubules are known to be involved in retinal mosaic formation , and tubulin regulation is important to zebrafish CNS regeneration and development . TH has been implicated in regulating tubulin in other neural regeneration paradigms .
TH has recently been found to effect the optical transparency of the rainbow trout lens, probably through modulation of ocular sodium/potassium ATPases  which are modulated by TH in other salmonid tissues . We detected three peptides of sodium-potassium ATPases (Table 3), none of which changed in abundance in an impressive manner.
Other known effects of TH in the retina include modulation of deiodinases  and changes in rhodopsin/porphyropsin ratios [27-30], although the pathways that regulate the latter, involving an unidentified 3,4-dehydrogenase, remain unknown. Two peptides similar to bacterial dehydrogenases, with unknown function in vertebrates, were increased threefold by TH; these might bear further exploration. We also know that TH affects opsin expression in the trout retina [12,14,17-20], discussed in the next section.
The peptides reported by the ICAT software for the LWS and MWS cone opsins are identical to the amino acid sequence predicted from our cDNA clones . Both opsins appear to be increased in abundance during TH treatment (Table 4). Similarly, both of the SWS2 opsin peptides are an exact sequence match to the protein predicted by our cDNA clone of the gene . The difference in the results from the SWS2-derived peptides (1.3 and 1.5 fold changes were observed) was relatively small. It is a matter of interpretation and further investigation whether this increase in SWS2 opsin is biologically significant. A recent examination as to opsin mRNA levels after nine days of TH treatment (identical to the current methods) using quantitative real-time RT-PCR (QPCR) supports the conclusion that our TH treatment affected cone opsin gene expression . The QPCR results are in accordance with those described here; the TH: Control ratio of transcripts was 0.77 for RH1, 2.1 for SWS2, and 1.7 for MWS (compare to Table 4). Only the ratio of LWS opsin transcripts differed from the current results, with TH having no apparent effect on LWS opsin transcripts.
Changes in visual sensitivity during TH treatment are also measurable and include a loss of sensitivity to UV light [13,14,17-19] and an increased sensitivity of the MWS cone mechanism , similar to results from natural development . The TH-induced increase in sensitivity of the MWS mechanism is consistent with the MWS opsin peptide being the most increased opsin in the current results, with a 2.5 fold increase in MWS opsin protein abundance.
Two peptides were detected from the rod opsin gene, RH1. One was a perfect sequence match to the RH1 gene product predicted from cDNA  and apparently changed little with TH treatment (ratio=0.98). The other peptide, which decreased in abundance with TH treatment (ratio=0.59), only matched the predicted gene product in 10 of the 12 amino acid residues. Together, these results suggest that a second RH1 gene is expressed in the rainbow trout retina, as is the case with some eels [88,89]. Indeed, another salmonid, the ayu (Plecoglossus altivelis) possesses two RH1 genes, although only one copy has been isolated in the retina at the developmental stages that were examined . Reduced RH1 expression (as measured by in situ hybridization) has been reported in early Xenopus embryos treated with TH and retinoic acid together, although not detectably with TH alone .
The only class of opsin not detected was SWS1 opsin, which is expressed in UVS cones [13,14]. It could be that there was effectively a complete lack of SWS1 opsin protein present after TH treatment. Our past analyses have detected minimal amounts of SWS1 opsin after TH treatment as measured by in situ hybridization and immunohistochemistry [12-14] as well as by QPCR . If this were the case, SWS1 would not be detected by ICAT because the technique relies on detecting doublets of peptides (separated by 9 Da) in the MS spectra. In this context one peptide is intriguing: It was identified by the software as a singleton in the control retina, and thus was not detected in the TH-treated retina. It was identified RH1 opsin from the European eel (Anguilla anguilla, see Table 4). The Oncorhynchus mykiss RH1 cloned previously  predicts 15 of 18 amino acids in the peptide reported; however, it lacks a cysteine residue in this region and cannot be the source of the peptide, as the ICAT label binds only to cysteine. The cysteine is also absent from this peptide in the RH1 opsins of other salmonids [79,92]. The SWS1 gene is the only opsin identified from trout that contains a cysteine in the 18 amino acids that share sequence identity with this peptide (SSCVYNPLIYAFMNKQFN). Perhaps a second SWS1 gene is expressed in the trout retina, similar to another salmonid . Thus, this peptide may be a portion of the missing SWS1 opsin present only in the control retina, or as mentioned previously, there may be another copy of the RH1 gene to which this peptide can be attributed.
Other photoreceptor-specific proteins were detected, including two peptides from arrestin, which were decreased 0.69 and 0.62 fold (Table 1, Table 3).
Several peptides were detected that play a role in retinal development in other model organisms. These include ΔA (matching the sequence from zebrafish Danio rerio ), which was less abundant (ratio=0.55) in TH-treated retina, and ΔC (matching the sequence of a cDNA clone from Atlantic salmon, Salmo salar), which was also less abundant (ratio=0.73) but did not meet our criterion. The Δ-Notch pathway is involved in retinal cell fate decisions [94,95] including the spatial patterning of cone photoreceptors in zebrafish . We also detected a peptide from Notch2 (matching the sequence from pufferfish, Takifugu rubripesi; Table B in Appendix 1), but no quantification was available.
We detected retinoic acid receptor gamma (RXR, matching the sequence from zebrafish Danio rerio) and found it did not change in abundance (ratio=0.85), similar to recent results in TH-treated Xenopus embryos . Retinoic acid and RXR are known to be involved in retinal development [97-100], including retinoic acid's effect on rainbow trout UVS cone ontogeny in a manner similar to TH .
We also detected a peptide from a protein with similarity to a GI:30154764 homeobox protein that increased almost 25 fold during TH treatment. Various homeobox genes have been shown to be involved in retinal development [101-105]. Also of note was a peptide from an inhibitor of metalloproteinase that increased 9.5 fold in abundance, and a metalloproteinase peptide (both matched the sequence of cDNA clones from Atlantic salmon Salmo salar) that did not change in abundance (ratio=0.979). Matrix metalloproteinases are known to be regulated by TH  and are involved in tissue remodeling including retinal development and disease [107-109].
Teleost retinas provide substantial advantages to understanding retinal development and function. The salmonid retina is a good example of this, with properties such as continued growth, regenerative capacity, and a highly ordered mosaic of photoreceptors (and other neurons) that facilitate a study of neuronal connectivity and interaction [110-112]. The salmonid retina has further interesting characteristics, including developmental plasticity of photoreceptors late in life history. This plasticity may be correlated with changes in the fish's environment, including migrations between deep-shallow or marine-freshwater habitats. Further, the large size of the salmonid eye compared to other popular teleost and rodent retinal models provides enough material for proteomic assessment of retinal development.
The approach we describe simultaneously identifies and quantifies the relative abundance of many proteins in two similar protein pools. By using commercially available facilities, one can quickly attain an overview of the retinal proteome and characterize changes in expression, even in rarely used animal models. Future work can help define criteria that establish guidelines for concluding that a given protein has changed in abundance. Like each high-throughput technique, repetition of results combined with confirmation through other approaches is required. The results of this study clearly demonstrate the potential of this technique to provide relevant and novel information in understanding retinal development and physiology.
We thank Professor Robert Olafson for helpful comments on the manuscript. Also, we thank Derek Smith and the University of Victoria Genome British Columbia Proteomics Center for their generous assistance. We are grateful for an Alzheimer Society/Canadian Institutes of Health fellowship (WTA) and an operating grant from the US Air Force Office of Scientific Research BioInspired Program (Grant number F49620-01-1-0506 PI: CWH). We thank the Vancouver Island Trout Hatchery for providing us with rainbow trout.
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