Proteomic Analysis of the Intestinal Adaptation Response Reveals

Nov 30, 2009 - Prince Henry's Institute of Medical Research, Monash Medical Centre, ... Gastroenterology and Clinical Nutrition, Royal Children's Hosp...
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Proteomic Analysis of the Intestinal Adaptation Response Reveals Altered Expression of Fatty Acid Binding Proteins Following Massive Small Bowel Resection Andrew N. Stephens,*,†,# Prue M. Pereira-Fantini,*,‡,# Guineva Wilson,§ Russell G. Taylor,§ Adam Rainczuk,† Katie L. Meehan,† Magdy Sourial,§ Peter J. Fuller,† Peter G. Stanton,† David M. Robertson,† and Julie E. Bines‡,|,⊥ Prince Henry’s Institute of Medical Research, Monash Medical Centre, Clayton, Murdoch Children’s Research Institute, Departments of Surgery and Gastroenterology and Clinical Nutrition, Royal Children’s Hospital, and Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia Received October 28, 2009

Intestinal adaptation in response to the loss of the small intestine is essential to restore enteral autonomy in patients who have undergone massive small bowel resection (MSBR). In a proportion of patients, intestinal function is not restored, resulting in chronic intestinal failure (IF). Early referral of such patients for transplant provides the best prognosis; however, the molecular mechanisms underlying intestinal adaptation remain elusive and there is currently no convenient marker to predict whether patients will develop IF. We have investigated the adaptation response in a well-characterized porcine model of intestinal adaptation. 2D DIGE analysis of ileal epithelium from piglets recovering from massive small bowel resection (MSBR) identified over 60 proteins that changed specifically in MSBR animals relative to nonoperational or sham-operated controls. Three fatty acid binding proteins (L-FABP, FABP-6, and I-FABP) showed changes in MSBR animals. The expression changes and localization of each FABP were validated by immunoblotting and immunohistochemical analysis. FABP expression changes in MSBR animals occurred concurrently with altered triglyceride and bile acid metabolism as well as weight gain. The observed FABP expression changes in the ileal epithelium occur as part of the intestinal adaptation response and could provide a clinically useful marker to evaluate adaptation following MSBR. Keywords: 2D-PAGE • DIGE • intestinal failure • MSBR • adaptation • short bowel syndrome • fatty acid binding protein • FABPL • FABP6 • FABPI

Introduction Short bowel syndrome (SBS) is a state of malabsorption and malnutrition, arising from either congenital disease or massive small bowel resection (MSBR), that results in a significant loss of functional intestinal length.1-3 In newborns and infants, the mortality associated with SBS can reach 90%, making it one of the most lethal conditions in infancy and early childhood.3-9 In a small number of SBS patients, the remaining functional bowel is unable to supply sufficient energy and nutrition requirements to maintain growthsa state known as intestinal failure (IF)sand the patient becomes dependent on long-term * To whom correspondence should be addressed. Dr. Andrew N. Stephens, Level 4, Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton VIC 3168, Australia. Phone +613 95947912. Fax +613 9594 7909. E-mail [email protected]. Dr. Prue Pereira-Fantini, Murdoch Children’s Research Institute, Parkville VIC 3052, Australia. Phone +613 83416452. Fax +613 8341-6449. E-mail [email protected]. † Prince Henry’s Institute of Medical Research. ‡ Murdoch Children’s Research Institute. § Department of Surgery, Royal Children’s Hospital. | Departments of Gastroenterology and Clinical Nutrition, Royal Children’s Hospital. ⊥ Department of Paediatrics, University of Melbourne. # These authors contributed equally to the manuscript. 10.1021/pr900976f

 2010 American Chemical Society

parenteral nutrition (PN) for survival.2,10 However, complications arising from the chronic administration of PN, such as bacterial sepsis and parenteral nutrition-associated liver disease (PNALD), are implicated in up to 5% of annual, nondisease related mortality and make long-term PN undesirable.2,3,7,11-13 Chronic PN also significantly alters quality of life and has high associated health-care costs.3,14 Bowel transplantation has been proposed as a preferred treatment for patients with IF,14-18 but the long-term requirements for immunosuppression coupled with a 5-year survival rate similar to chronic PN currently make transplantation unsuitable for most IF patients.3,15,16,19 Following a significant loss of mucosal surface after MSBR, the remaining intestine must undergo compensatory physiological and morphological changes to increase its absorptive capacity.10,20 This process of intestinal adaptation, involving compensatory growth and the dilation, thickening, and lengthening of remaining intestine, can be detected almost immediately and may last for a period of months to years.3,10,20,21 The adapting intestine undergoes prominent hyperplasia and hypertrophy in all layers of the bowel wall, a process primarily driven by intestinal epithelial stem cells within the intestinal crypts.22 Increased epithelial cell proliferation and migration along the crypt-villus axis results in lengthening of the villi, Journal of Proteome Research 2010, 9, 1437–1449 1437 Published on Web 11/30/2009

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Stephens et al. 10,11,23-26

deepening of the crypts and thickening of the mucosa. This is accompanied by alterations in the rate of apoptosis and an increase in cellular DNA, RNA and protein content.3,10,20 Concomitant changes in nutrient uptake, ion transport and digestive capacity ideally lead to increased absorption of carbohydrate, protein, water and electrolytes per unit length in the small intestine,3,11,25,26 although absorption per cell is decreased due to a lack of mature cells in the villi.10 These changes are most prominent in the ileum and are directly proportional to the amount of bowel remaining.3,10 The success of the adaptive response directly determines whether a patient will regain enteral autonomy or progress to a state of intestinal failure. In those patients where IF is irreversible and long-term dependence on PN is considered life-threatening, intestinal transplantation is indicated as the most appropriate treatment.2,14-18 The key to successful transplantation lies with early referral, prior to the development of life-threatening complications and the requirement for simultaneous multiorgan transplant.2,17-19 It is therefore imperative that patients who will not achieve enteral autonomy are distinguished as soon as possible, to facilitate the best long-term management and patient prognosis.3,11 However, to date there is no clinically useful marker with sufficient accuracy to predict at an early stage whether patients will develop IF. A number of factors are known to influence the success of intestinal adaptation and have been extensively reviewed; these include the composition of luminal nutrients, pancreaticobiliary secretions, secreted hormones and requirement for peptide growth factors.2,3,8,10,13,27-30 Several studies have attempted to characterize gene expression following MSBR to analyze mechanisms of intestinal adaptation,11,20,21,31 or to characterize the developmental progression of proliferating cells migrating along the crypt-villus axis.32 However the mechanisms underlying the successful intestinal adaptation response, including the molecular signals that initiate, maintain and ultimately terminate the adaptation process, remain poorly understood.20,21,31 A marker of intestinal adaptation would be of significant clinical use, aiding in the evaluation of patient response to therapeutic interventions that may promote intestinal adaptation as well as allowing for earlier referral of patients for transplantation who will not achieve ultimate enteral autonomy. We have applied a proteomic strategy to analyze protein expression in a well-established porcine model of intestinal adaptation33 following MSBR. This is the first study describing the application of proteomic technology to investigate the changes in protein expression that occur during adaptation of the intestinal lumen as a result of SBS.

Experimental Section Animals. All experiments were conducted according to the guidelines of the National Health and Medical Research Council, and with prior ethics approval from the Animal Ethics Committee of the Murdoch Children’s Research Institute (Melbourne, Australia). Weaned 3-week old Landrace/Large White Cross piglets (Victorian Institute of Agricultural Science, Werribee, Australia) were acclimatized at the Royal Children’s Hospital Animal Research Laboratory (Melbourne, Australia) at a constant temperature of 22 °C with a 12 h light cycle for one week prior to surgery. Surgical procedures have been described previously.33-36 In brief, 4-week old piglets underwent either a 75% proximal small bowel resection (MSBR), transection and reanastomosis (SHAM), or no operation (NOC). 1438

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Small bowel resection included removal of the small bowel from 1 m distal to the ligament of Treitz to 2.5 m proximal to the ileocecal valve. The SHAM operation group were transected and reanastomosed 2.5 m proximal to the ileocecal valve. Following surgery all animals (both control and treatment groups) received identical peri- and postoperative care. Animals were maintained in individual cages to accurately measure diet consumption. Weight was measured weekly prior to the morning feed. Serum required for fasting bile acid, cholesterol and triglyceride measurements was collected 24 h prior to operation and at 2, 4, and 6 weeks postoperatively. Samples were evaluated in the Department of Laboratory Medicine (Royal Children’s Hospital, Parkville, Australia) using established methodology. After sacrifice ileal tissue was harvested, and either placed in 10% neutral buffered formalin (Australian Biostain Pty. Ltd., Australia) for paraffin embedding or immediately snap frozen. Prior to protein extraction the ileal mucosa was manually separated from the underlying muscle. Expression Analysis and Protein Identification. Protein extraction from snap-frozen ileal tissue, fluorescent protein labeling using CyDyes and two-dimensional polyacrylamide gel electrophoresis (2D DIGE) were as described.37 Labeling was performed using 50 µg protein as recommended by the manufacturer. Isoelectric focusing was carried out using both pH 3-10 and pH 5-8 gradients according to the following parameters; 60 µA per strip, 100 V/90 min, 300 V/90 min, 500 V/3 h, gradient to 1000 V/4 h, gradient to 8000 V/3 h, constant 8000 V until reaching 60 000 Vh. Differential expression analysis based on normalized spot volumes was carried out using PG240 Same Spots software (Nonlinear Dynamics, Newcastle-uponTyne, UK). All proteomic analysis was carried out on individual animal samples in each group. Reciprocal labeling experiments were also carried out using a pooled protein sample, and any proteins showing differential labeling effects specific to the use of the Cy3/Cy5 dyes were eliminated from the analysis. Protein spots of interest were excised using a ProPicII robotic spot picker (Genomic Solutions, MI) based on the X-Y coordinates exported directly from PG240 SameSpots. Protein identification by MALDI-TOF MS and MS/MS was also as described.37 Monoisotopic peak masses were automatically extracted using GPS Explorer software (v 3.0 build 311; Applied Biosystems, CA) and peak lists searched against the nonredundant UniProtKB/Swiss-Prot database (release 57.3; 468851 sequence entries; http://www.uniprot.org) using the MASCOT search engine (updated 03-01-2007; http://www.matrixscience.com). Species was restricted to mammalian, carbonylamide-cysteine (CAM - fixed modification) and oxidation of methionine (variable modification) were taken into account, a parent ion mass tolerance of 0.1 Da and 1 missed cleavage (enzyme specificity trypsin) was allowed. Up to fifteen of the most intense peptides detected in each MS scan were automatically selected for MS/MS analysis. Peak lists were extracted using Data Analysis software version 3.4 (Bruker Diagnostics, Germany). The parameters used to create the peak lists were as follows: mass range 100 to 3000 Da; signal-to-noise threshold of 5; minimum compounds length of 10 spectra. Combined peptide mass (MS) and fragmentation (MS/MS) data were searched using in-house MASCOT search engine (version 1.1, Matrix Science) against the UniProtKB/Swiss-Prot database as above, with fragment mass tolerance of 0.1 Da. The following criteria were used to evaluate the search; MOWSE score (e44 for PMF data; g 72 for combined MS/MS data), number of peptides matched (g6 for PMF data; g2 for combined MS/MS

Fatty Acid Binding Protein Expression from Intestinal Adaptation

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Figure 1. Characterization of dietary intakes in piglets following surgery. (A) Energy and (B) total weight gain were measured in piglets over the 6-week experimental period. NOC, nonoperational controls; SHAM, sham-operated controls; MSBR, massive small bowel resection group. Mean ( SEM; n ) 6/group.

data); and the estimated molecular mass and pI as determined from the 2D gel. Statistical Analysis. All statistical analyses of proteomic expression data were performed automatically by the PG240 SameSpots software. Expression data are given as mean normalized spot volume ( standard deviation. Hierarchical clustering and protein expression heat maps were generated using open source R software (http://www.r-project.org/). Statistical analysis of immunoblotting and immunohistochemistry results was carried out using SigmaPlot software (Systate Software Inc., Chicago, IL) with data expressed as mean ( standard error of the mean (SEM). Immunoblotting. Total protein was extracted from snapfrozen, unfixed ileal tissue using TRIzol reagent (Invitrogen, CA) according to the manufacturer’s instructions. Protein pellets were resuspended in 0.1% SDS (w/v) and protein content determined using the bicinchononic acid (BCA) protein assay kit (Pierce Biotechnology, IL). A 10 µg aliquot of protein from each specimen was separated in a 10% SDS PAGE tris-glycine gel, and transferred to nitrocellulose membrane (GE, Uppsala, Sweden). Proteins were detected using polyclonal antibodies directed against FABP-6 (1:2000; R&D Systems, Minneapolis, MN), I-FABP (1:3000; R&D Systems, Minneapolis, MN) and L-FABP (1:10 000; Abcam, Cambridge UK) or β-actin (Sigma Aldrich, USA) and a secondary HRP-conjugated antibody directed against either goat or rabbit as required. ECL reagent (GE Healthcare) was used to visualize protein detection, and images were analyzed using ImageJ software. All quantitation was performed relative to a β-actin control. Immunohistochemical Staining. Immunohistochemical staining was carried out on paraffin-embedded, formalin-fixed sections of ileal tissue. Monoclonal antibodies directed against FABP-6 (1:50; R&D Systems, Minneapolis, MN), I-FABP (1:100; R&D Systems, Minneapolis, MN) and L-FABP (1:100; Abcam, Cambridge UK) were used to visualize protein localization. Sections were photographed and the number of positively stained cells in either the crypt or villus quantitated using ImageJ software (v1.34s; http://rsb.info.nih/gov/ij). All image analysis was performed by an experienced histologist. Biological Enrichment and Pathway Analysis. Proteins identified as differentially expressed following MSBR, relative to both SHAM and NOC controls, were further analyzed for enrichment of specific biological processes and pathways using GeneGO pathway analysis software (Version 4.3, Build 9311; http://www.GeneGO.com). For each identified protein, the appropriate human homologue was first located in the UniProtKB/Swiss-Prot database using the online Protein Identifier Cross-Reference Service (http://www.ebi.ac.uk/Tools/picr/ search.do).38 The accession numbers of these human homo-

logues were then uploaded into the GeneGO environment along with specific proteomic fold change data. Pathway analysis and GO ontologies were determined using the manually curated MetaCore database. Ranking of relevant pathways and GeneGO processes was based on hypergeometric distribution p values as performed by the software, with a p value of e10-6 considered significant.

Results Recovery from Surgery and Characterization of the Adaptive Response in MSBR Piglets. To evaluate the recovery of animals following surgery, the nutrient intake and weight gain of piglets from the NOC, SHAM, and MSBR groups were monitored over a 6 week recovery period. During the initial 2 weeks following surgery, piglets in the MSBR group had a reduced energy intake compared to piglets from either of the SHAM or NOC groups; by postoperative week 4, however, the energy intake of all animals was comparable (Figure 1a). Prior to surgery all animals were of comparable weight, with piglets that underwent MSBR initially showing a reduced rate of weight gain compared to NOC and SHAM animals (Figure 1b). While the absolute weight of animals in the MSBR group remained lower than that of animals in the NOC or SHAM groups, by postoperative week 5 the rate of weight gain in each group was similar (Figure 1b), indicating that animals that had undergone MSBR made a successful recovery following surgery. Following surgical bowel resection there is a diminished capacity of the remaining bowel to absorb lipids and reclaim luminal bile acids.39 Therefore, the levels of serum bile acids, triglycerides, and cholesterol were assessed throughout the postoperative period. Neither serum bile acid nor triglyceride levels altered significantly in SHAM-operated animals compared to the NOC group (Figure 2a,b). By contrast, serum bile acid and triglyceride levels were significantly increased in the MSBR animals relative to both SHAM and NOC groups (Figure 2a,b; serum bile acids: p < 0.01 vs NOC, p < 0.05 vs sham; serum triglyceride: p < 0.001 vs. NOC, p < 0.01 vs sham). There was no significant difference in serum cholesterol levels observed between any of the experimental groups (Figure 2c). Next, a microscopic evaluation of the intestinal villi and crypt surface areas was undertaken to determine whether morphological changes in the intestinal mucosa, characteristic of intestinal adaptation, had occurred in the MSBR group. Animals in both the SHAM and NOC groups displayed no apparent difference in the total villus or crypt area after the 6 week recovery period; by contrast, both crypt and villus areas were increased in MSBR animals (Figures 3a and b). Lengthening of the villi and deepening of the intestinal crypts is a wellJournal of Proteome Research • Vol. 9, No. 3, 2010 1439

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Figure 4. Representative 2D PAGE images showing differentially expressed proteins following MSBR. Isolated proteins from NOC, SHAM and MSBR animals were analyzed by 2D DIGE in both pH 3-10 (A) and pH 5-8 (B) gradients. Significantly (p e 0.01) differentially expressed proteins in MSBR animals relative to both NOC and SHAM groups are indicated. Table 1. Summary of Proteomic Expression Data Following Intestinal Adaptationa

Figure 2. Bile acids, triglyceride and cholesterol levels in piglets following surgery. Total serum levels of bile acids, triglycerides and cholesterol were measured in piglets throughout the 6 week recovery period following surgery. NOC, nonoperational controls; SHAM, sham-operated controls; MSBR, massive small bowel resection group. Mean ( SEM; n ) 6/group. t test; *p e 0.05; **p e 0.01, ***p e 0.001.

Experiment pH 3-10 Total # spots observed Total differences observed increased in MSBR vs NOC decreased in MSBR vs NOC increased in MSBR vs SHAM decreased in MSBR vs SHAM Biological variability NOC within group (% CV) SHAM MSBR

1223 71 58 13 48 11 26% 30% 23%

Experiment pH 5-8 Total #spots observed Total differences observed increased in MSBR vs NOC decreased in MSBR vs NOC increased in MSBR vs SHAM decreased in MSBR vs SHAM Biological variability NOC within group (% CV) SHAM MSBR

1533 53 35 18 30 15 25% 31% 24%

a Isolated ileal tissue from animals that underwent either no operation (NOC), control intestinal resection and anastomosis (SHAM) or 75% massive small bowel resection (MSBR) were compared by 2D DIGE across pH 3-10 and pH 5-8 gradients. Differentially expressed proteins were deemed to be those showing significant (p e 0.01; ANOVA) expression changes of g1.5 fold between the groups. Biological variability between individuals in each group was assessed as average CV for all protein spots resolved in each data set.

Figure 3. Changes in crypt and villus area in piglets following MSBR. Total (A) crypt and (B) villus area in isolated segments of ileal tissue was measured in NOC (nonoperational controls), SHAM (sham-operated controls) and MSBR (massive small bowel resection) piglet groups. Mean ( SEM; n ) 6/group. t test; ***p e 0.001.

characterized adaptive response following surgery.10,11,23-26 Taken together, the data indicate that piglets in the MSBR 1440

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group displayed intestinal adaptation following a 75% loss in small intestine length. Proteomic Expression Analysis Reveals Specific Changes in MSBR Animals Reflecting Intestinal Adaptation. Ileal tissue from the NOC, SHAM and MSBR groups was extracted and the protein expression compared by 2D DIGE across both pH 3-10 and pH 5-8 gradients (Figure 4a and b). Proteins displaying significant expression changes in MSBR animals (p e 0.01; ANOVA) of greater than 1.5-fold compared to either the NOC or SHAM groups were considered to be differentially expressed. The results of the two individual proteomic expression analyses are summarized in Table 1. A total of 71 and 53 significant differences between the three groups were observed across the pH 3-10 and 5-8 ranges, respectively (Table 1). The majority of these represented increased protein expression levels in MSBR animals relative to both of the NOC and SHAM groups. Of these differences, 59 (pH 3-10), and 45 (pH 5-8), were significantly altered in MSBR animals relative to both of the

Fatty Acid Binding Protein Expression from Intestinal Adaptation

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proteins have been proposed as a potential link between intestinal resection, successful adaptive response and PNassociated liver disease. Three of the fatty acid binding proteins identified by proteomic analysissliver fatty acid binding protein (L-FABP), intestinal fatty acid binding protein (I-FABP) and gastrotropin (FABP-6)swere independently identified in both pH 3-10 and pH 5-8 proteomic profiling experiments. We therefore chose to concentrate on the expression changes observed for these proteins.

Figure 5. Unsupervised hierarchical clustering and expression mapping of proteins observed in proteomic profiles. Logtransformed normalized protein spot volumes were used to perform unsupervised hierarchical cluster analysis. Green indicates decreased expression; red indicates increased expression. Operation group (NOC, SHAM or MSBR) and pH range is indicated.

SHAM and NOC controls (Table 1). These proteins, representing expression changes specific to MSBR animals, were flagged for subsequent analyses. Biological variation between individuals within each group, as assessed by coefficient of variance (CV) in normalized protein spot volumes, was consistently between ∼20-30% across the two experiments (Table 1) indicating good reproducibility within the model. Unsupervised hierarchical cluster analysis was then conducted using log normalized protein expression levels from each proteomic data set. In each case, animals that underwent MSBR consistently clustered into a single discrete group, based on protein expression, relative to both SHAM and NOC animals (Figure 5a and b). By contrast, SHAM and NOC animals were not separated from each other suggesting that the control surgical procedure involving transaction and reanastomosis of the small intestine (SHAM) did not significantly affect protein expression associated with intestinal function relative to the nonoperated controls (NOC). Together, this demonstrates that significantly altered protein expression in the ileum following MSBR could clearly discriminate animals undergoing intestinal adaptation from nonresected animals. Differentially expressed proteins were then submitted for identification by mass spectrometry. Of a total of 107 protein spots analyzed (including 3 additional spots that were significantly different between MSBR and NOC, but not SHAM, animals), 63 were unambiguously identified and a further 20 were reported as mixtures of 2 or more proteins, representing an overall total of 62 unique proteins (Table 2). Twelve proteins were independently identified in both pH 3-10 and pH 5-8 profiling experiments, while a further 24 protein spots (16 from pH 3-10; 8 from pH 5-8) remained unidentified and were not examined further. Validation of Fatty Acid Binding Protein Expression and Localization. In the context of intestinal adaptation, fatty acid metabolism is of particular interest as the FABP family of proteins are key modulators of intracellular nonesterified fatty acids, bile salts and the bile acid pool. In particular, these

To validate the expression changes observed by 2D DIGE, immunoblotting was carried out using antibodies against L-FABP, FABP-6 and I-FABP (Figure 6). In each case, immunoblotting was performed on isolated ileal tissue from 6 animals in each group. Increased total expression of both L-FABP (Figure 6a; right panel) and FABP-6 (Figure 6b; right panel) was observed by immunoblot in MSBR animals relative to both NOC and SHAM controls, confirming the changes observed in proteomic expression data (Figure 6a and b; lefthand panel). By contrast, I-FABP showed no change in MSBR animals relative to NOC (Figure 6c; right panel), contradicting the changes observed by proteomic profiling (Figure 6c; lefthand panel). Therefore, changes in total expression of L-FABP and FABP-6, but not I-FABP, were confirmed in MSBR animals following the 6 week recovery period. To further characterize the effects of MSBR on intestinal FABP expression, immunohistochemical staining against each of the FABP’s was carried out on ileal tissue sections from NOC, SHAM and MSBR animals and the number of positively stained cells within both the crypt and villus quantified (Figure 7). Since the crypt drives cell renewal within the intestine, changes in cell numbers within the crypt reflect influences on cell growth whereas changes in villus cell numbers reflect increased absorption and intestinal function. MSBR animals displayed increased numbers of L-FABP-positive cells within both the crypts and villi compared to SHAM and NOC control groups (Figure 7a), again confirming the increase in total L-FABP expression. By contrast increased numbers of FABP-6-positive cells were observed in the villi of MSBR animals, but not in the crypts (Figure 7b) compared to both SHAM and NOC. This suggests that either site-specific expression changes or altered FABP-6 localization has occurred within the intestinal epithelium following adaptation. No significant change in either total expression or localization of I-FABP was observed in MSBR animals relative to NOC controls (Figure 7c). Biological Expression Profiling and Enrichment Analysis. To identify key molecular and biological functions that were overrepresented within the data set, all proteins identified were analyzed using GeneGO biological pathway profiling software. Enrichment analysis was first performed to identify differentially regulated canonical pathways that were over-represented by the proteins involved in intestinal adaptation. Five welldefined canonical pathways were identified as significantly enriched within the data set (Table 3; also see Supplementary Data S1). Of these, four specifically related to the regulation of cytoskeletal remodelling via actin, tubulin and keratin intermediate filaments (pathways 1, 2, 4 and 5; Table 3) and involved key pathways mediating intracellular cytoskeletal rearrangements.40 The fifth pathway identified involved increased mitochondrial oxidative phosphorylation. These biological processes would be expected of adapting small intestine, which undergoes significantly increased cell proliferation and cytoskeletal rearrangements relative to nonadapting intestine.26 Journal of Proteome Research • Vol. 9, No. 3, 2010 1441

identified

ACTB_CANFA ANXA4_PIG APOA4_PIG APOA4_PIG ARP3_HUMAN ARP3_HUMAN ATP5H_BOVIN ATPA_MOUSE ATPB_HUMAN ATPB_RAT ATPB_RAT ATPB_RAT ATPB_RAT CALX_RAT CO6A3_HUMAN COF1_HUMAN COF2_HUMAN DERM_BOVIN DERM_PIG EF1A1_CRIGR EF2_BOVIN EF2_PONAB EF2_PONAB FABP6_PIG FABPI_PIG FABPL_PIG FABPL_PIG FETUA_PIG GANAB_PIG GDIB_PIG GRP78_BOVIN GRP78_BOVIN GRP78_HUMAN GRP78_HUMAN GRP78_HUMAN HS90A_BOVIN HSPB1_PIG K1C20_PIG K1C20_PIG K1C20_PIG KCY_PIG MLRN_HUMAN MVP_HUMAN PARK7_BOVIN PDIA1_BOVIN PDIA1_MACFU PDIA1_MOUSE PDIA3_HUMAN PDIA4_HUMAN

protein spot #

163 69 52 123 84a 192a 240 227a 148 87 158a 160 229 93a 63 11b 11b 20 7 227a 191 221 223 394 5 1 6 93a 194a 177a 92a 120 113 128 174 124 239a 84a 177a 192a 239a 115 194a 239a 43 93a 362 207 92a

1442

Q6QAQ1 P08132 O46409 O46409 B5APU4 none listed none listed none listed none listed Q29235 (fragment) Q29235 (fragment) none listed none listed Q29160 (fragment) none listed none listed Q5G6 V9 P45846 P45846 none listed none listed none listed none listed P10289 Q45KW7 P49924 P49924 P29700 P79403 Q6Q7J2 P34935 P34935 (fragment) P34935 P34935 P34935 none listed Q5S1U1 Q29218 Q29218 Q29218 Q29561 P29269 none listed Q0R678 none listed none listed none listed none listed none listed

UniPROT acc. (Sus scrofa) ACTB ANXA4 APOA4 APOA4 ACTR3 ACTR3 ATP5H ATP5A1 ATP5B ATP5B ATP5B ATP5B ATP5B CANX COL6A3 CFL1 CFL2 DPT DPT EEF1A1 EEF2 EEF2 EEF2 FABP6 FABP2 FABP1 FABP1 AHSG GANAB GDIB HSPA5 HSPA5 HSPA5 HSPA5 HSPA5 HSP90AA1 HSPB1 KRT20 KRT20 KRT20 CMPK1 MYL9 MVP PARK7 P4HB P4HB P4HB PDIA3 PDIA4

gene

MOWSE score

From pH 3-10 Profiling Actin, cytoplasmic 1 610 Annexin A4 1010 Apolipoprotein A-IV 92 Apolipoprotein A-IV 52 Actin-related protein 3 64 Actin-related protein 3 121 ATP synthase subunit d, mitochondrial 135 ATP synthase subunit alpha, mitochondrial 78 ATP Synthase subunit beta, mitochondrial 429 ATP synthase subunit beta, mitochondria 1460 ATP synthase subunit beta, mitochondria 171 ATP Synthase subunit beta, mitochondrial 140 ATP Synthase subunit beta, mitochondrial 141 Calnexin 76 Collagen alpha-3(VI) chain 107 Cofilin-1 263 Cofilin-2 139 Dermatopontin 136 Dermatopontin 96 Elongation factor 1-alpha 1 132 Elongation factor 2 639 Elongation factor 2 169 Elongation factor 2 267 Gastrotropin 580 Fatty acid-binding protein, intestinal 638 Fatty acid-binding protein, liver 414 Fatty acid-binding protein, liver 347 Alpha-2-HS-glycoprotein 146 Neutral alpha-glucosidase AB 146 Rab GDP dissociation inhibitor beta 181 78 kDa glucose-regulated protein 319 78 kDa glucose-regulated protein 492 78 kDa glucose-regulated protein 1280 78 kDa glucose regulated protein 171 78 kDa glucose-regulated protein 1380 Heat shock protein HSP 90-alpha 214 Heat shock protein beta-1 235 Keratin, type I cytoskeletal 20 224 Keratin, type I cytoskeletal 20 218 Keratin, type I cytoskeletal 20 109 UMP-CMP kinase 74 Myosin regulatory light polypeptide 9 405 Major vault protein 155 Protein DJ-1 334 Protein disulfide-isomerase 478 Protein disulfide-isomerase A1 133 Protein disulfide-isomerase 386 Protein disulfide-isomerase A3 99 Protein disulfide-isomerase A4 153

protein

Table 2. Differentially Expressed Proteins Identified by MALDI-TOF Mass Spectrometry

11 24 13 8 10 5 3 10 20 18 14 16 15 3 15 11 7 2 3 10 23 13 19 12 11 6 6 3 15 7 17 20 34 19 31 14 8 8 9 5 7 8 13 7 13 10 13 8 15

no. peptides identified 33.3% 61.5% 26.2% 22.0% 29.9% 12.7% 24.2% 17.2% 48.2% 42.0% 36.1% 19.8% 40.8% 5.1% 4.6% 53.0% 34.3% 9.5% 11.5% 18.4% 24.2% 14.3% 21.3% 75.0% 60.6% 46.5% 46.5% 7.7% 15.4% 18.4% 27.6% 32.4% 54.6% 42.5% 50.6% 20.7% 40.6% 57.0% 74.8% 35.5% 39.3% 44.8% 14.8% 40.7% 26.7% 20.2% 25.0% 15.2% 17.7%

sequence coverage % 41.7/5.29 35.8/5.71 43.3/5.69 43.3/5.69 47.3/5.61 47.3/5.61 18.7/5.59 59.7/9.22 56.3/5.19 56.3/5.19 56.3/5.19 56.3/5.19 56.3/5.19 67.2/4.49 343.3/6.4 18.5/8.22 18.7/7.66 24.0/4.86 22.0/4.86 50.1/9.1 95.3/6.41 95.3/6.41 95.3/6.41 14.2/6.84 15.2/6.62 14.1/6.59 14.1/6.59 38.4/5.5 106.6/5.64 50.2/6.31 72.4/5.07 72.4/5.07 72.3/5.07 72.4/5.07 72.3/5.07 84.7/4.93 22.9/6.23 12.5/8.8 12.5/8.8 12.5/8.8 22.2/6.02 19.8/4.8 99.3/5.64 20.0/6.84 57.2/4.8 57.3/4.69 57.1/4.79 56.7/5.98 72.9/4.96

mass/pI 1.6 1.9 2.0 1.0 1.9 1.6 1.5 1.5 1.0 1.9 1.7 1.0 1.0 1.8 1.6 -2.7 -2.7 -2.3 -2.2 1.5 1.5 1.5 1.5 1.7 3.1 3.3 2.9 1.8 1.6 1.6 1.8 1.7 1.7 1.1 1.6 1.7 -1.5 1.9 1.6 1.6 -1.5 -1.7 1.6 -1.5 2.1 1.8 1.7 1.5 1.8

fold change vs NOC 1.7 1.5 2.0 1.7 1.6 1.5 1.5 1.5 1.6 1.6 1.6 1.7 1.5 1.8 2.0 -2.3 -2.3 -2.5 -2.9 1.5 1.6 1.5 1.5 1.2 2.1 2.4 2.1 1.8 1.5 1.5 1.5 1.6 1.6 1.7 1.5 1.7 -1.5 1.6 1.5 1.5 -1.5 -1.7 1.5 -1.5 1.9 1.8 1.8 1.6 1.5

fold change vs SHAM 9.33 × 4.19 ×