Identifying Candidate Serum Biomarkers of Exposure to Tunicamycins in Rats Using Two-Dimensional Electrophoresis Megan A. S. Penno,*,†,‡ Antony Bacic,§ Steven M. Colegate,‡ Peter Hoffmann,† and Wojtek P. Michalski‡ Adelaide Proteomics Centre, University of Adelaide, Adelaide, South Australia 5005, Australia, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, Victoria 3220, Australia, and Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia Received December 24, 2008
A model for chronic corynetoxins poisoning has been established in rats exposed to the toxicologically bioequivalent inhibitor of N-linked glycosylation the tunicamycins. Consumption of corynetoxins in contaminated pasture can result in the often fatal neurological disease of grazing livestock annual ryegrass toxicity (ARGT). Corynetoxins may also threaten human health as potential contaminants of the food supply via grain or products derived from subclinically exposed animals. The serum proteomes of four dose groups plus a control group following 6, 9, and 12 months dietary tunicamycins exposure were compared by one-dimensional electrophoresis. Numerous differences were observed between the control and the highest dose group (40.5 µg tunicamycins/kg of body weight/day), designated as High). Accordingly, these samples were further examined using two-dimensional electrophoresis. Thirtythree protein spots were found to be differentially displayed between the Control and High dose sera based on univariate statistics (p < 0.05 for log10 transformed normalized volumes) and significant foldchanges in spot volume ((2.3-fold as determined by posthoc power analysis). Identities for 28 spots were obtained by MALDI-TOF MS corresponding to 13 different proteins. An increasing population of carbohydrate deficient transferrin was identified in the High dose sera using a combination of antibody and lectin detection and confirmed by ESI-IT MS/MS. The functionalities of other identified proteins were consistent with the oxidative stress and acute phase responses. The biomarkers identified in this study may not only play a useful role in diagnosing toxin exposure but could be helpful in identifying new treatment strategies for ARGT and equivalent human diseases. Keywords: Biomarkers • two-dimensional electrophoresis • toxicology • tunicamycins/corynetoxins • annual ryegrass toxicity
Introduction Toxicology in combination with proteomics seeks to identify critical proteins and pathways that are affected by the chemical stimuli using techniques for protein expression analysis.1 One of the biological fluids frequently used in proteomic studies is blood serum, which is predicted to contain in excess of 10 000 000 structurally different protein species with a dynamic range of 10-15 orders of magnitude.2,3 Consequently, the serum proteome has been described as the single most informative sample that describes the current state of health of an individual.1 In this study, a proteomic strategy involving serum was employed to identify candidate biomarkers of annual ryegrass toxicity (ARGT), a disease of significant importance to Australia’s agricultural industries and potentially to human health. * To whom correspondence should be addressed. E-mail: Megan.Penno@ adelaide.edu.au. † University of Adelaide. ‡ Australian Animal Health Laboratory, CSIRO Livestock Industries. § University of Melbourne.
2812 Journal of Proteome Research 2009, 8, 2812–2826 Published on Web 03/24/2009
The severe neurological manifestations of ARGT, which predominantly affects sheep and cattle, are caused by the ingestion of the naturally occurring tunicaminyluracil glycolipid toxins, the corynetoxins (CTs).4-6 Other members of the tunicaminyluracil-based family include the tunicamycins (TMs), streptovirudins, antibiotic 24010, MM 19290 and the mycosporcidins.7 Each of these toxin families share a common N-acetyl glucosaminyltunicaminyluracil (GTU) core structure linked to a fatty acid side chain.8 There are up to 12 congeners in the group of CTs, differing in the length, presence of hydroxylation or unsaturation, and terminal branching structure of the fatty acid chain. While some CTs overlap with the family of TMs, the fatty acid chains on the TMs are usually shorter and are not hydroxylated compared to the CTs.9 Several in vivo studies have shown that exposure to TMs produces a syndrome that is clinically and pathologically indistinguishable from ARGT.10-12 Consequently, due to their commercial availability, TMs have been used to model ARGT in experimental animals. CTs are produced by the bacterium Rathayibacter toxicus following the colonization of annual ryegrass (Lolium rigidum) 10.1021/pr801111a CCC: $40.75
2009 American Chemical Society
Biomarkers of Tunicamycins Exposure seed heads via the expediency of the Anguina funesta nematode (reviewed in ref 13). Like the well-characterized TMs, CTs are potent irreversible inhibitors of the highly conserved liver enzyme N-acetylglucosamine-1-phosphate transferase (GPT).12 The exquisite sensitivity of GPT to the toxins can be measured via an activity assay using liver biopsy samples.14,15 Although the assay provides a dose-dependent indicator of exposure, it is unsuitable for the routine testing of livestock due to the invasive and expensive nature of sample collection. At present, there are no simple, noninvasive diagnostic tests for detecting CTs exposure. GPT catalyzes the first step of oligosaccharide biosynthesis in the N-glycosylation pathway. It has been suggested that the depletion of N-glycan side chains as a result of GPT inhibition and consequent impairment of N-linked glycoprotein production may be responsible for ARGT.16 A comprehensive review of the pathology associated with acute CTs poisoning has been published previously.13 The effects associated with low-level exposure to CTs, however, are not well-understood. Both published data and anecdotal claims have indicated that nonfatal, and even subclinical exposure to CTs in sheep can affect wool growth17 and reproduction.18,19 Studies in rats have also shown that a single subclinical dose of TMs can lead to male infertility19 and that pregnant rats are more susceptible to TMs than nonpregnant cohorts.20 Exposure to CTs is also a suspected issue for human health as there is an unquantified potential for the contamination of the human food supply. This may occur directly via contaminated grain or indirectly from food products derived from subclinically exposed animals. While there have been no known cases of human disease attributed to CTs, their acute toxicity and cumulative nature necessitates the development of a detection assay, particularly at chronic levels of CTs-exposure, that would be suitable for both livestock and humans.21 The primary biochemical effect of TMs and CTs exposure involves the inhibition of GPT and subsequent depletion of N-glycosylated proteins. In this study, a biomarker discovery strategy based on proteomics was employed to identify serum proteins that may change (i.e., through differential expression, aberrant post-translational modification, etc.) following longterm low-level exposure of rats to TMs.
Experimental Section Animals. Experimental procedures involving animals were performed in compliance with approved protocols and ethical standards set by the Australian Animal Health Laboratory’s Animal Ethics Committee (AEC Protocol 879). Six-week old outbred Hooded Wistar rats (University of Adelaide, SA, Australia) were divided into five groups containing seven males and seven females such that the mean body weights of each group were equal. In these groups, the rats were exposed to 0 (Control), 1.5 (Very Low), 4.5 (Low), 13.5 (Medium) or 40.5 (High) µg/kg body weight/day of TMs in their pellet food for up to 12 months. TMs were spiked into American Institute of Nutrition 93 semipurified rodent diet pellets22 manufactured by Specialty Feeds (Glen Forrest, WA, Australia). The dosages were calculated based on a pilot study in which growth rates and food intakes were measured and were intended to be spread across a range from a possible no observed effect level (NOEL) to a level where adverse effects were detected. For quality control purposes, the levels of TMs in the pellets were confirmed by ELISA as described by Than et al.23 (results not shown). Rats were housed in same-sex pairs and maintained at a constant
research articles 23 °C with a 12-h light/dark cycle. Access to reverse osmosis deionized water was provided ad libitum and all were monitored by a veterinarian daily as stipulated by the AEC. Serum Collection. Whole blood was collected from CO2 anesthetized rats via cardiac puncture and immediately transferred to Capiject T-MG serum collection tube (Terumo Medical Corporation, Somerset, NJ). The blood was allowed to clot for 1 h at 37 °C and centrifuged at 1000g for 5 min to obtain the serum. Sera were aliquoted into 50 µL volumes and stored at -80 °C. Pooled samples were prepared by combining equal volumes of serum from animals belonging to the same gender, dose-group and time point. One-Dimensional Electrophoresis (1-DE). Serum samples were diluted at 1/1000 in MOPS buffer (50 mM MOPS, 50 mM Tris base, 3.5 mM SDS, 1 mM EDTA, pH 7.7), then mixed with 4× lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA) and 0.5 M DTT in a 5:2:1 volume ratio, respectively. Electrophoresis was performed using NuPAGE 4-12% Bis-Tris polyacrylamide gels (Invitrogen) run at a constant 180 V. Gels were visualized by silver staining according to the PlusOne Silver Staining Protocol for Proteins (GE Healthcare, Uppsala, Sweden) with the omission of glutaraldehyde from the sensitizing solution. Analytical 2-DE. Seven microliters of pooled serum was diluted in 43 µL of ultrapure water (18.2 MΩ/cm), reduced with 10 µL of 150 mM DTT, heated at 95 °C for 5 min to ensure complete reduction,24 then diluted with 40 µL of ultrapure water. Once cooled, 12 µL of sample was combined with 488 µL of rehydration buffer (8 M urea, 2% CHAPS, 65 mM DTT, 0.5% 3-10 NL IPG buffer (GE Healthcare) and a trace amount of bromophenol blue for color). One-hundred and twenty-five microliter aliquots of the 500 µL samples were applied to four 7 cm 3-10 NL IPG strips (GE Healthcare) via active rehydration loading in ceramic strip holders (GE Healthcare). Isoelectric focusing was carried out using an IPGphor I (GE Healthcare) at 20 °C using a stepwise gradient of 12 h at 50 V, 1 h at 500 V, 1 h at 1000 V and up to 12 h at a maximum of 8000 V until 27 000 Vh total had been achieved. The current was limited at 50 µA per strip. Immediately following IEF, the strips were equilibrated in reducing and alkylating buffers as recommended by the manufacturer. The equilibrated strips were run on Novex 4-20% Tris-Gly Zoom gels (Invitrogen) at a constant 180 V. The resulting gels were stained with silver nitrate as described above and digitally scanned (Umax, Taipei, Taiwan). Preparative 2-DE. Ten microliter amounts of serum from the five 12-month Control males and five 12-month High dose males were pooled. The 100 µL pooled sample was reduced with 23 µL of 150 mM DTT, heated at 95 °C for 5 min, cooled to room temperature, diluted with 204 µL of 3% CHAPS, and mixed with 163 mg of urea. Next, 1.7 µL of 3-10 NL IPG buffer and 23.7 µL of 1 M DTT were added plus a trace amount of bromophenol blue for color. A 340 µL aliquot of the prepared sample was applied to an 18 cm 3-10 NL IPG strip (GE Healthcare) via active rehydration in a ceramic strip holder. IEF was carried out using an IPGphor II (GE Healthcare) at 20 °C using the following program: 12 h at 50 V, 1 h at 500 V, 1 h at 1000 V and 5 h at a maximum of 8000 V until 40 000 Vh total had been achieved. The current was limited at 50 µA per strip. Following IEF, the equilibrated strip was run on a 12.5% polyacrylamide gel (25 cm × 20 cm × 0.1 cm) cast using the EttanDalt 12 gel casting system (GE Healthcare) according to the manufacturer’s recommendation with glass plates treated with 4 mL of Bind silane solution (80% ethanol, 2% acetic acid, Journal of Proteome Research • Vol. 8, No. 6, 2009 2813
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0.1% Bind silane; GE Healthcare). Electrophoresis in the second dimension was performed using an EttanDalt 12 separation unit in Tris-gly buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 15 °C at a constant 75 V for 16 h. The gel, attached to the glass plate, was fixed for 1 h (40% ethanol, 10% acetic acid) then stained with Coomassie Brilliant Blue (8% ammonium sulfate, 0.8% phosphoric acid, 0.08% CBB G-250, 20% methanol). Spots within the preparative gel could be easily matched to the analytical gels based on the resulting patterns and positions of the molecular weight markers. Image Analysis. Scanned images of the analytical 2-DE gels were exported into ImageMaster 2-D Elite version 4.01 (GE Healthcare/Nonlinear Dynamics, Newcastle, U.K.). Spots were manually detected and matched to the master gel, which was an artificial representation of all of the spots identified across the 48 gels in the experiment. Comparative statistics were performed on the volumes of the valid matched spots (i.e., spots detected in at least three of the quadruplicate gels within a group, or in at least two gels of one group where three or four spots were detected in the corresponding group). The unprocessed spot volumes were exported into Excel (Microsoft, Redmond, WA) and normalized to express each spot’s volume as a percentage of the total volume of all the valid spots on the given gel. The normalized volumes (NV) were log10 transformed (log10NV) to normally distribute the data and enabled parametric statistical tests to be performed.25,26 The log10NV values of the matched Control and High dose spots for each time and gender cohort were compared using a two-tailed, unpaired, Student’s t test. The fold-change in a spot’s volume was calculated by dividing the average NVHigh by the average NVControl for a given time and gender cohort. If the resulting value was less than 1, the value was divided into -1, thereby reflecting a negative fold-change in the High dose relative to the Control group. Those spots that were identified only within the High dose data set were given the arbitrary fold-change value of 10. Those spots that were found to be absent from the High dose group (i.e., Control-only) had a corresponding fold-change of -10. Power analysis was used to calculate the effect size (and subsequent fold-change) at which the mean NVs of matched Control and High dose spots could be considered significantly different. This was performed using the online power calculator Piface (version 1.64).27 The required information to identify the effect size was the sample size, the level of power, the significance level and the standard deviation. The standard deviations of the log10 NV within each time, gender and dose group were calculated in Excel. The following argument was used to translate the effect size to a fold-change (based on the equations of Karp and Lilley26): log10 NVHigh - log10 NVControl ) effect size log10(NVHigh /NVControl) ) effect size NVHigh /NVControl ) 10effect size
(1)
fold change ) 10effect size To evaluate the possibility that a candidate may constitute a false positive two additional tests were performed. First, the relative expression levels of the spots in the High dose serum were assessed for dose dependency across the time course of the experiment. At the three time point the NVs of each spot across the quadruplicate High dose male and High dose female gels were averaged and compared. Second, to evaluate the impact of multiple hypothesis testing, the false discovery rate (FDR) in the form of a q-value was determined for each 2814
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matched spot based on the log10 NV p-values calculated using the software tool QVALUE (version 1.0).28 In QVALUE, lambda was set at 0.5 and the smoothing method was used to determine π0, which represented the proportion of features that are unchanging.29 In-Gel Tryptic Digestion. The spots of interest were excised from the preparative 2-DE gel using an Ettan Spot Cutting Robot (GE Healthcare). The spots were destained and digested with 100 ng (10 ng/µL in 5 mM ammonium bicarbonate) of sequencing grade modified trypsin (Promega, Madison, WI) per sample. The resulting tryptic peptides were extracted from the gel pieces with 50% acetonitrile (ACN), 0.3% formic acid (FA) in water. The volumes of the final samples were reduced from approximately 120 µL to approximately 1 µL by vacuum centrifugation. The peptides were then diluted to approximately 5 µL with FA30 (7 parts 0.1% FA, 3 parts ACN). MALDI-TOF/TOF MS. One microliter of each sample was applied to a 600 µm AnchorChip target (Bruker Daltonics, Bremen, Germany) in a matrix of R-cyano-4-hydroxycinnamic acid (CHCA; Bruker Daltonics) according to the manufacturer’s thin layer method. MALDI-TOF mass spectra were acquired using an Ultraflex II MALDI TOF/TOF mass spectrometer (Bruker Daltonics) operating in positive ion reflectron mode under the control of the FlexControl software (Version 3.0, Bruker Daltonics). External calibration was performed using peptide standards (Bruker Daltonics) that were analyzed under the same conditions. MALDI-TOF spectra were obtained from random positions on the anchor spot at a laser intensity determined by the operator to produce optimal resolution. A selected number of the most highly abundant ions were subjected to MS/MS analysis. These MALDI-TOF/TOF spectra were acquired in LIFT mode using the same spot on the target. The MS and MS/MS spectra were analyzed using FlexAnalysis software (Version 3.0, Bruker Daltonics). Ion detection was performed using the SNAP algorithm with a signal-to-noise threshold of 3, a quality factor threshold of 50 and the number of detected peaks limited to 1000 for MS and 300 for MS/MS spectra. The MS mass lists were exported to BioTools (Version 3.1, Bruker Daltonics) and submitted to an in-house licensed version of the Mascot database-searching engine (version 2.2, Matrix Science, Boston, MA). The search parameters were the following: taxonomy ) Rattus (67 940 sequences), database ) NCBInr (updated on 20/12/2007), enzyme ) trypsin, global modifications ) carbamidomethylation of cysteine, variable modifications ) oxidation of methionine, MS mass tolerance ) 100 ppm, missed cleavages ) 1. Proteins that returned MOWSE scores above the threshold of 61 were considered to be significant hits. If a protein identification was made a mass control list was created based on masses of the expected tryptic peptides of the protein and the spectra were internally recalibrated in FlexAnalysis. If a preliminary identification was not made, the spectra were internally calibrated against the masses of the m/z 842.509 and m/z 2211.104 trypsin autolysis peaks. The recalibrated MS mass lists and corresponding MS/MS spectra were exported to BioTools, combined, and submitted to Mascot. The search parameters were as described above with the following exceptions: MS mass tolerance ) 25 ppm, MS/ MS mass tolerance ) 0.5 Da. The annotated MS spectra and sequence coverage diagrams for each protein identified within a spot are presented in Supporting Information Figure S1. A list of the peptides for which MS/MS data were obtained is
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Biomarkers of Tunicamycins Exposure presented in Supporting Information Table T1. The threshold ion score for a single MS/MS spectrum was 28. LC-ESI-IT MS. Following an in-gel tryptic digest, the peptide samples were reconcentrated from 5 µL to approximately 0.5 µL by vacuum centrifugation, diluted with 4 µL of 0.1% FA, and transferred to autosampler vials. An Agilent Technologies (Santa Clara, CA) 1100 HPLC system equipped with a nanoflow pump, capillary pump, micro autosampler and 10-port valve was used for the separation of tryptic peptides. Two microliters of the samples was injected via the auto sampler and loaded on a Zorbax 300SB-C18, 3.5 µm, 5 × 0.3 mm trap column (Agilent Technologies) at a flow rate of 10 µL/min in mobile phase A (3% ACN, 0.1% FA in water) by the capillary pump. After 10 min, a 10-port valve was switched automatically and the nanoflow pump delivered mobile phase B (0.1% FA in ACN) as follows: 3-40% gradient over 40 min, 40-60% gradient over 5 min, held at 60% for 3 min, 60-80% gradient over 7 min, held at 80% for 2 min, reduced from 80 to 3% over 3 min, then held at 3% for 13 min. The peptides eluting from the trap column were separated on the analytical column (Zorbax 300SB-C18, 3.5 µm, 150 × 0.075 mm; Agilent Technologies) at a flow rate of 0.2 µL/min. The online column was coupled via a distal-coded micro tip needle (10 µm internal diameter) into a high capacity 3-D IT mass spectrometer (HCT ultra, Bruker Daltonics). The HPLC-separated peptides were trapped and the two most intense ions were fragmented by CID. Active exclusion was used to exclude a precursor ion for 30 s following the acquisition of two spectra. MS and MS/MS spectra were subjected to peak detection and deconvolution using DataAnalysis (Version 2.4, Bruker Daltonics). The MS and MS/MS mass lists were exported to BioTools then submitted to Mascot (version 2.2). The specifications were taxonomy ) Rattus (67 940 sequences), database ) NCBInr (updated on 13/10/ 2007), enzyme ) trypsin, fixed modifications ) carbamidomethylation of cysteine, variable modifications ) oxidation of methionine, peptide mass tolerance ) (0.3 Da, fragment mass tolerance ) (0.4 Da, missed cleavages ) 1, peptide charge ) 1+, 2+ and 3+. MS/MS spectra were annotated in BioTools based on the Mascot results. Western and Lectin Blotting. Serum samples separated by 1-DE (as above) were electrotransferred onto BioTrace PVDF membrane (Pall Life Science, Pensacola, FL) at 170 mA for 1 h using a Bio-Rad Transblot apparatus (Hercules, CA) in 10 mM CAPS, 10% (v/v) methanol (pH 11) transfer buffer. When performing immunodetection, PVDF membranes were blocked overnight at 4 °C in skim milk and probed with a polyclonal rabbit-anti-rat transferrin primary antibody (MP Biomedicals, Irvine, CA), and a goat-anti-rabbit alkaline phosphatase conjugated secondary antibody (Chemicon, Temecula, CA). The blots were developed with nitro blue tetrazolium chloride/5bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt (NBT/ BCIP) (Roche, Basel, Switzerland) containing 0.05 M MgCl2, 0.1 M NaCl in 0.1 M Tris-HCl, pH 9.5. When performing lectindetection, PVDF membranes were blocked overnight with Blocking Reagent supplied with the Digoxigenin Glycan Differentiation Kit (Roche, Basel, Switzerland). Membranes were probed with a biotinylated Sambucus nigra agglutinin (SNA) lectin (Vector Laboratories, Burlingame, CA) followed by streptavidin-horseradish peroxidase conjugate (Calbiochem, Darmstadt, Germany). Enhanced chemiluminescence detection of the lectin blots was performed using the ECL+plus Western Blotting Detection System (GE Healthcare) in conjunction with ECL Hyperfilm (GE Healthcare) as directed by the manufacturer.
Results and Discussion Clinical and Physiological Effects in Rats. In this study, male and female rats were exposed to four levels of dietary TMs, a known inhibitor of protein N-glycosylation, for up to 12 months. The dosages were intended to be spread across a range of potential outcomes from a possible NOEL to a level at which adverse effects would be detectable. A Control group received exactly the same handling, feed and water but without TMs. The rats showed no dietary aversion to the TMs-spiked pellets as evidenced by the long-term constancy (i.e., over several months) of body weight/feed intake ratios and by separate short-term studies over several weeks that compared intake of TMs-spiked and unspiked pellets by rats of both genders (Colegate, personal communication). No clinical effects of the dietary TMs were observed in any of the groups throughout the dosing period; thus, the candidate biomarkers identified in the presented study are, by definition, preclinical. Histologically, hepatic changes indicative of mild liver damage in the High dose males after 12 months were observed (not shown). By contrast, at 12 months, the residual liver GPT activities of the TMs exposed rats were seriously affected as described by Stewart et al.30 As very little is known regarding long-term low-level CTs exposure in the field, it is difficult to determine whether the in vivo dose rates reported here represent a plausible scenario in the field. Outbreaks of ARGT resulting from exposure to heavily contaminated oaten hay have been reported.31 Therefore, it is conceivable that livestock could be progressively exposed to chronic levels of CTs through fodder that was harvested from a mildly toxic pasture without showing outward signs of disease. Biomarker Discovery Strategy. After 6 and 9 months of the study, four animals from each dose groups (two male, two female) were euthanized and serum was collected to provide some time-course data. The only exception to this was the 9 month Low dose male group, which had only one biological replicate due to the premature death of its cage-mate (presumably unrelated to toxin exposure). After 12 months of the study, to provide more statistically significant data, 10 animals from each group (five male, five female) were euthanized and serum was collected. At each time point, the serum samples from the same gender and dose groups were pooled and initially analyzed by 1-DE. Relative to the Control groups, considerable changes were observed only in the High dose groups at 6, 9, and 12 months for both males and females (Figure 1). Given that the High dose rats have received an approximate total of 7.2 mg/kg of body weight of TMs after 6 months while the Medium dose group was exposed to only 4.8 mg/kg of body weight by 12 months, the minimum amount of toxin required to cause visible changes to the proteome must be between these concentrations. Only one gender-related change was observed. Males showed a band at approximately 80 kDa, whereas females showed a band at approximately 75 kDa (Figure 1). The identities of these proteins are unknown, but they do not appear to be related to or affected by the treatment. To further investigate the apparent treatment-related differences in the gel profiles, pooled sera from the Control and High dose groups for males and females at each time point were analyzed by 2-DE. Gels were run in quadruplicate to compensate for potential gel-to-gel variation.32 All the 48 gels within the experimental dataset showed well-separated spot patterns for proteins with observed molecular weight (MW) Journal of Proteome Research • Vol. 8, No. 6, 2009 2815
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Figure 1. The 1-DE analysis of pooled male and female serum samples taken from rats exposed to TMs for 6, 9, and 12 months. The regions highlighted by the arrows show differences between the Control through to the Medium and High dose profiles. The asterisk indicates a region of the gel that differs between the pooled male and female samples. Numbers on the left indicate the molecular weight markers.
values of less than 100 kDa (Supporting Information Figure S2). The high MW proteins (i.e., >100 kDa) did not resolve as well, although the streaking patterns that were generated were fairly uniform across the all gels of the experiment (refer to Supporting Information Figure S2). The overloading of albumin was necessary to visualize the proteins of lower abundance, although horizontal streaking occurred near the site of albumin’s MW of approximately 66 kDa. A depletion strategy for the major serum proteins including albumin may have further enriched the lower abundance species. However, depletion was not used in this study as it was predicted that the highly abundant proteins, particularly those that are glycosylated (including transferrin, haptoglobin, fibrinogen and R1-antitrypsin, which are removed using rodent-specific immunoaffinity columns33), would be clinically relevant in this disease model given the toxin inhibits N-linked glycosylation. Furthermore, it has been reported that only a small number of new proteins are detected on a 2-DE gel following depletion of rat plasma.33 More sophisticated fractionation strategies involving up to four dimensions of separation may enable deeper mining of the proteome;34 however, these were unavailable in the laboratory at the time of the experiment. Visual inspection of the analytical 2-DE gels revealed a number of differences between the Control and High dose gels for males and females at the 6, 9, and 12 month time points (highlighted in Supporting Information Figure S2). The average numbers of valid spots detected on the quadruplicate gels for each gender, time and dose group are presented graphically in Figure 2A. At all time points, there were more spots present on the High dose than the Control serum gels for both males and females. Although marginal, this increase was found to be statistically significant in five of the six gender/time pairs (p < 0.05). Comparative Analysis of Spot Volumes. The statistically acceptable magnitude of the fold-change between Control and 2816
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High dose spots was determined using posthoc power analysis. A power calculation of this type, which determines the effect size (i.e., the size difference between means), takes into consideration the amount of variance in the data set, the required significance level, the sample size and the desired level of power.26 The effect size was calculated by inputting the standard deviation value that encompassed 75% of the log10 NV data (calculated to be 0.17), the sample size (four, since analyses were in quadruplicate), the target power level (0.8), and a significance level of 0.05 into the calculation tool Piface. Substitution of the derived effect size of 0.3618 into eq 1 determined that a fold-change level of 2.3 would indicate a significant difference in spot volume. Because a pooling strategy was used, the experimental variance included in the power calculation refers to the amount technical variation between the replicate gels and not biological variation. Although some suggest that pooling can overcome background variation attributed to genetic differences between animals and thus is acceptable, even advisable,32 others have reported that pooling serum samples may be associated with a significant loss of potential biomarkers.35 In the presented study, it was unpractical to analyze all biological replicates in this experiment using single-sample gels. While a multiplexed approach such as the DIGE platform36 would have been ideal for this experiment, the technology is very expensive and was inaccessible to our laboratory. Accordingly, a pooled sample strategy was used with future plans to validate the expression of the candidate biomarkers in individual rat serum samples. For a matched spot to be considered as a candidate biomarker of toxin exposure, it was required to (i) have p-values of 2.3 in at least three of the six time and gender cohorts, and (ii) show a similar pattern of regulation in at least 4 of the 6 cohorts. Thirty-three spots fulfilled these criteria. The individual fold-changes of the candidates are provided graphically in Supporting Information
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Figure 2. (A) The numbers of valid spots detected on the Control and High dose gels (averaged from the four gels) prepared from male and female rat sera following 6, 9, and 12 months exposure to TMs (mean ( standard deviation). A Student’s t test was performed to compare the numbers of spots on the Control and High dose gels within the same time and gender groups. The resulting p-values are indicated on the graph. (B) The positions of the identified candidate biomarker spots on a representative 2-DE gel from a 12 month High dose male rat serum, and (C) 12 month Control rat serum. The quadruplicate gels from these and all other dose groups are presented in Supporting Information Figure S2. The spots highlighted in red had consistently higher spot volumes in the Control rats throughout the experiment (i.e., were down-regulated in the High dose). Spots highlighted in green had higher volumes in the High dose data set (i.e., up-regulated in High dose). Spots highlighted in dark blue were detected only in the High dose groups for males and females at all time points. Spots highlighted in light blue were not detected in the Controls at any time point, but were found in High dose gels at 4-5 time points. Those spots that showed mixed spot volumes are highlighted in yellow (refer to Supporting Information Figure S3). The arrows show the positions of transferrin with a distinct lower train in the High dose gel. The numbers on the left-hand side of the gels indicate the molecular weight markers. Journal of Proteome Research • Vol. 8, No. 6, 2009 2817
research articles Figure S3 and summarized in Figure 2B,C. Here, the relative positions of the 33 candidates on a representative High dose and Control gel (Figure 2, panels B and C, respectively) are shown. Nine spots were consistently identified only in the High dose male and female rat sera at all time points (spots 334, 335, 336, 351, 352, 358, 359, 362, and 363: Figure 2B,C, dark blue circles). Spots 360 and 361 were identified as High-only in five of the six cohorts, and spots 408, 409, and 410 were identified as High-only in four cohorts (Figure 2B,C, light-blue circles). This reflected the results shown in Figure 2A where significantly more spots were detected in the High dose gels compared to the Controls. Eight spots were generally up-regulated in the High dose rats and were, in certain cohorts, identified only in the High dose group (spots 227, 230, 340, 341, 349, 369, 403, 407: Figure 2B,C, green circles). Six spots were consistently down-regulated in the High dose rats relative to the Controls (spots 72, 211, 312, 313, 314, 315: Figure 2B,C, red circles). The remaining five spots showed mixed patterns of regulation between the Control and High dose group across the time spans (spots 29, 38, 153, 344, 347: Figure 2B,C, yellow circles). The 33 candidates were further ranked as Class I, II, or III candidates according to their likely biological relevance based on two independent criteria: the q-value and dose dependency (Figure 3A). Spots classified as Class III candidates showed a dose dependent increase or decreased in relative abundance based on the High dose NV of the spot at each time point (averaged for males and females, Figure 3B) and had an average q-value
