Temporal Proteomic Analysis Reveals Continuous Impairment of Intestinal Development in Neonatal Piglets with Intrauterine Growth Restriction Xiaoqiu Wang,† Weizong Wu,† Gang Lin,† Defa Li,†,* Guoyao Wu,†,‡ and Junjun Wang*,† State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China 100193, and Department of Animal Science, Texas A&M University, College Station, Texas 77843 Received September 7, 2009
Efficiency of nutrient utilization is reduced in neonates with intrauterine growth restriction (IUGR) compared with those with a normal birth weight (NBW). However, the underlying mechanisms are largely unknown. In this study, we applied temporal proteomic approach, coupled with histological and biochemical analyses, to study dynamic changes of the proteome in the small intestinal mucosa of IUGR piglets during the nursing period (Days 1, 7 and 21). We identified 56 differentially expressed protein spots between IUGR and NBW piglets. These proteins participate in key biological processes, including (1) absorption, digestion and transport of nutrients; (2) cell structure and motility; (3) glucose and energy metabolism; (4) lipid metabolism; (5) amino acid metabolism; (6) mineral and vitamin metabolism; (7) cellular redox homeostasis; (8) stress response; and (9) apoptosis. The results of our temporal proteomics analysis reveal continuous impairment of intestinal development in neonatal piglets with IUGR. The findings have important implications for understanding metabolic defects in the small intestine of IUGR neonates and are expected to provide new strategies to improve their survival and growth. Keywords: Intrauterine growth restriction • Piglets • Intestine • Development • Temporal proteomic analysis • Histological analysis
Introduction Genetic, epigenetic, maternal maturity, and environmental factors (e.g., maternal nutrition, heat stress, disease, and toxins) are important factors affecting fetal growth and development.1 Intrauterine growth restriction (IUGR) is defined as impaired growth and development of the mammalian embryo/fetus or its organs during pregnancy, which can be measured as fetal or birth weight less than two standard deviation of the mean body weight for gestational age.2 Fetal growth retardation is a significant problem in both humans3 and livestock species.4 Of particular interest, the pig exhibits the most severe naturally occurring IUGR among mammalian species.4 IUGR is a major factor contributing to high neonatal mortality because of impaired development of the small intestine.5,6 The small intestine is the major organ for terminal digestion and absorption of nutrients.7 The gut is also a defense barrier against diet-derived pathogens, carcinogens and oxidants.8 Additionally, in mammals, the small intestine is the exclusive organ for endogenous synthesis of citrulline (from glutamine/ glutamate and proline) and arginine,9 which has versatile roles * To whom correspondence may be addressed. Dr. Defa Li or Dr. Junjun Wang: State Key Laboratory of Animal Nutrition, China Agricultural University, No. 2. Yuanmingyuan West Road, Beijing, China, 100193. Phone: +8610-62733588. Fax: +86-10-62733688. E-mail:
[email protected] or
[email protected]. † China Agricultural University. ‡ Texas A&M University.
924 Journal of Proteome Research 2010, 9, 924–935 Published on Web 11/26/2009
in growth, development and health.10 Results of our recent study with newborn pigs indicate that IUGR negatively affects expression of proteins in the small intestine of piglets at birth that are related to cellular signaling, redox balance, protein synthesis, and proteolysis.5 At present, little is known about the effects of IUGR on the intestinal proteome during postnatal development. In the swine industry, the first postnatal week is the most critical period for neonatal survival,4 and piglets are usually weaned at 21 days of age to increase the productivity of sows.11 Therefore, the objective of this study with the piglet model was to quantify temporal changes in small-intestinal proteins between days 1 and 21 of life.
Materials and Methods Piglet Model and Tissue Collection. During the entire period of gestation, gilts (Large White sires × Landrace dams; n ) 18 litters) were fed 2 kg/day of a corn and soybean meal-based diet and had free access to drinking water, as we described previously.5 Eighteen litters of piglets (Large White × Landrace × Pietran) were spontaneously delivered from sows at term (day 114 of gestation). At birth, 1 IUGR piglet (∼ 0.7 kg) and 1 normal-birth-weight (NBW; ∼ 1.3 kg) piglet were obtained from each of 18 litters. The selected piglets (n ) 36; 18 IUGR vs 18 NBW) were positioned in the second teat pairs (known as anterior mammary glands)12 sucking milk from their own mother for 21 days. On day 0 (D0, without sucking milk), body weights of all neonatal piglets were recorded immediately upon 10.1021/pr900747d
2010 American Chemical Society
Impairment of Intestinal Development in Neonatal Piglets birth. On day 1 (D1), 7 (D7), and 21 (D21), neonatal piglets (6 IUGR and 6 NBW piglets) from each of 6 litters were weighed and then killed by jugular puncture after anesthesia, as we described previously.5 The small intestine in neonatal pigs was defined as the portion of the digestive tract between the pylorus and the ileocecal valve, with the first 10-cm segment being duodenum, and the subsequent 40 and 60% of the small intestine length below the duodenum being jejunum and ileum, respectively.5 The content of whole jejunum was rapidly removed with saline.13 After measuring the length and weight of the whole jejunum, approximately a 20-cm segment of midjejunum (the middle portion of jejunum) was obtained. A 3-cm portion of jejunum was fixed in 4% formaldehyde (Sigma, St. Louis, MO) at 4 °C for histological analysis and scanning electron microscopy (for Day 1 samples only). Mucosa from the remaining jejunal segment was obtained as described previously,13 rapidly placed in liquid nitrogen and stored at -80 °C for proteomic and Western blot analyses. The animal use protocol was reviewed and approved by the China Agricultural University Animal Care and Use Committee. Histology Analysis of Villus Morphology. For light microscopy, the formaldehyde fixed samples were embedded in paraffin, sectioned and mounted on glass slides for staining with hematoxylin and eosin, as we described.5 Intestinal morphology was examined with a light microscope (Olympus BX50, Japan). The villus height and width was quantified using the Medical Image Analysis System (MIAS) software. A minimum of 15 well-oriented, intact villi were measured in triplicate for each biological sample. For scanning electron microscopy, the formaldehyde-treated samples were fixed with 2.5% glutaraldehyde (Sigma, St. Louis, MO) in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature. They were then washed three times for 30 min with the same buffer, placed in 1% osmium tetroxide (Sigma, St. Louis, MO) for 1 h, and then washed again using the above procedures. The fixed samples were subsequently dehydrated in a graded ethanol series (30, 50, 70, 80, 90, 95, and 100%). The samples were transferred to isoamyl acetate (Sigma, St. Louis, MO), dried in a critical point drier (HITACHI HCP-2, Japan) before being coated with gold. The specimens were examined by scanning electron microscopy (HITACHI S-570, Japan). Protein Extraction of Jejunal Mucosa. Proteins were extracted from the jejunum mucosa as we described.14,15 Briefly, jejunal mucosa samples were homogenized in a lysis buffer (7 M Solid Urea, 2 M Thiourea, 4% CHAPS, 50 mM dithiothreitol (DTT)) containing 1% protease inhibitors (100×) (GE Healthcare, Piscataway, NJ). The protease inhibitor was specifically developed for sample preparation in two-dimensional electrophoresis studies to inhibit calpain II, cathepsin B, elastase, papain, plasmin, thermolysin and trypsin. Tissues were ruptured at 0 °C using an Ultrasonicater Model VCX 500 (Sonics & Materials, Newtown, CT) at 20% power output for 10 min with 2-s on and 8-s off cycles. After adding 1% (v/v) nuclease mix (GE Healthcare, Piscataway, NJ), the lysed cell suspension was kept at room temperature for 1 h to solubilize proteins,16 followed by resonication as described above to thoroughly break up cell membranes. The homogenate was subsequently centrifuged for 10 min at 13 000g at 15 °C. The supernatant fluid was collected, and its protein concentration was determined using a PlusOne 2-D Quant Kit (GE Healthcare, Piscataway, NJ). Protein extracts were stored in aliquots (1 mg of protein) at -80 °C.
research articles Two-Dimensional Gel Electrophoresis (2-DE). With one gel for each IUGR and NBW paired sample in each of the 3 timephases (D1, D7 and D21), a total of 18 gels were run for the 2-DE using commercial IPG strips (pH 3-10 NL, 24 cm) (GE Healthcare, Piscataway, NJ) for isoelectric focusing (IEF) and then standard vertical SDS-PAGE (12.5%) for second dimension. Briefly, mucosal extracts (1 mg protein/sample) were loaded onto IPG DryStrips using the in-gel sample rehydration technique, according to the manufacturer’s instructions.16 After rehydration for 12 h, the first-dimensional IEF was carried out at 20 °C for 100 000 Vh in the Ettan IPGphor II IEF system (GE Healthcare, Piscataway, NJ), as we described.17 Sequentially, IPG strips were equilibrated for 15 min in 4 mL of equilibration buffer-1 (6 M urea, 1% DTT, 30% glycerol, and 50 mM Tris-Cl pH 8.8) and then in 4 mL of equilibration buffer-2 (6 M urea, 2.5% iodoacetamide, 30% glycerol, 50 mM Tris-Cl pH 8.8) for 15 min. The second dimension was carried out on an Ettan DALT six (GE Healthcare, Piscataway, NJ) at 30 mA/gel for 30 min, and then at 50 mA/gel for about 6 h. In the seconddimensional procedure, temperature was set at 10 °C. The gels were then stained with colloidal Coomassie Brilliant Blue G-250 (Amresco, Inc., Solon, OH). Image Analysis. High-resolution gel images (600 dpi) were obtained using an ImageScanner Model PowerLook 2100XL (UMAX Technologies, Atlanta, GA) and image analysis was performed using an Image-Master 2D Platinum Version 6.01 according to manufacture’s protocol (GE Healthcare, Piscataway, NJ). After normalizing the quantity of each spot by total valid spot intensity, differentially expressed protein spots (P < 0.05) with a deviation of over 1.5-fold in the relative volume (% vol) were selected and subjected to identification by mass spectrometry (MS). In-Gel Digestion. Protein spots of interest were manually obtained and destained with 100 µL of 50% (v/v) acetonitrile (ACN) in 25 mM ammonium bicarbonate for 1 h. After the protein samples were completely dried by vacuum centrifugation (Eppendorf Concentrator 5301, Germany) for 30 min, they were digested with 2 µL of trypsin (Amresco, Inc., Solon, OH) in 25 mM ammonium bicarbonate at 4 °C for 1 h, and then incubated at 37 °C for 12 h. The resulting peptides were subjected to sequential extraction (3 times at 37 °C) with 8 µL each of 5% trifluoroacetic acid (TFA) for 1 h, 2.5% TFA in 50% ACN for 1 h, and 100% ACN for 1 h. Extracted protein samples were dried in a vacuum centrifugation. Protein Identification by MS and Database Search. Peptides from in-gel digested proteins were mixed with a matrix solution [R-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA, and 50% ACN]. MALDI-TOF/TOF MS (Matrix Assisted Laser Desorption Ionization-Time Of Flight/Time Of Flight MS) for protein identification was carried out on a 4700 MALDI-TOF/ TOF Proteomics Analyzer (Applied Biosystems, Foster City, CA) with 355-nm neodymium-doped yttrium aluminum garnet (Nd: YAG) laser and 20-kV accelerated voltage. After MS, 20 parent mass peaks with 700-3200 Da of mass range and minimum signal/noise values were selected for MS/MS analysis. Protein identification was achieved through Peptide Mass Fingerprint (PMF) and MS/MS data searches using GPS Explorer Workstation (Applied Biosystems, Foster City, CA) with the in-house searching engine Mascot and the searching taxonomy of Mammalia against the NCBInr database. Search parameters included: (1) trypsin, as the enzyme of protein digestion; (2) monoisotopic, as mass value; (3) unrestricted, as peptide mass; (4) (0.3 Da, as peptide mass tolerance; (5) oxidation (M) and Journal of Proteome Research • Vol. 9, No. 2, 2010 925
research articles
Wang et al.
Table 1. Body Weights of IUGR and NBW Piglets at Birth and during the Suckling Perioda
piglet
body weight at birth (n ) 18 per group) (kg)
IUGR 0.73 ( 0.01** NBW 1.31 ( 0.02
b
body weight (kg) day 1
day 7
day 21
0.88 ( 0.02** 1.94 ( 0.04** 5.13 ( 0.16** 1.46 ( 0.05 2.64 ( 0.06 6.52 ( 0.19
a Values are means ( SEM, n ) 6 per group except for body weight at birth. b **, P < 0.01 vs the NBW group.
carbamidomethyl (C), as variable modifications; and (6) 1, as maximum missed cleavages. In this procedure, a protein match with a score >71 was considered significant (P < 0.05). Western Blotting for Protein Analysis. Extracted proteins (30 µg/sample) were separated by electrophoresis (Bio-Rad, Richmond, CA) on 12.5% SDS-PAGE before being transferred electrophoretically to a PVDF membrane (Millipore, Billerica, MA). After blocking with TBST (0.05% Tween 20, 100 mM TrisHCl and 150 mM NaCl, pH 7.5) containing 5% fat-free dry milk at 4 °C overnight, the membranes were incubated with primary antibodies, that is, anti-ALB, anti-APOA1, anti-GRP94 and antiPRDX1 (Beijing Biosynthesis Biotechnology Co., Ltd., China) in dilution of 1:300, 1:300, 1:500 and 1:500, respectively for 2 h. The membranes were then rinsed in TBST and incubated with a secondary antibody (horseradish peroxidase-labeled antirabbit IgG diluted 1:1000) for 2 h. The protein bands were visualized with a chemiluminescence subtract using a gelimaging system (Tanon Science and Technology, Shanghai, China) with Image Analysis Software (National Institutes of Health, Bethesda, MD). Statistical Analysis. Normality of the data was tested using the Shapiro-Wilk test in SAS (version 8.1; SAS Institute, Cary, NC). Data were analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA, with each animal as an experimental unit. All analyses were performed using SAS. Data are expressed as means and SEM P < 0.05 was considered statistical significance.
Results Body Weights of Piglets. Body weights of IUGR and NBW piglets at birth (D0) were 0.73 and 1.31 kg, respectively (P < 0.01; Table 1). Between D1 and 21 of life, IUGR piglets continued to have a lower (P < 0.01) body weight than NBW piglets (Table 1). At D21, the body weight of IUGR piglets was 27% lower (P < 0.01) than that of NBW piglets. Jejunal Lengths and Weights. Table 2 summarizes the jejunal length and weights of IUGR and NBW piglets between D1 and 21 of life. The absolute jejunal length of IUGR piglets was shorter (P < 0.01) than that of NBW piglets, at each of 3 time-phases (D1, D7 and D21). Likewise, absolute jejunal weights and body weights of IUGR piglets were lower (P < 0.01) compared with NBW piglets at all time points. Compared with NBW piglets, the relative length of jejunum (jejunal length/ body weight) in IUGR piglets transitioned from a higher (P < 0.01) value on D0 to no difference (P > 0.05) on D7 and to a lower (P < 0.01) value on D21. However, the relative weight of jejunum was consistently lower (P < 0.01) in IUGR than in NBW piglets at all time points. Jejunal Villus Morphology. At D1, the villus height and width of jejunum were lower (P < 0.05) in IUGR than in NBW piglets (Table 3). The difference in jejunal villus height between IUGR and NBW piglets was even more pronounced (P < 0.01) at D21 926
Table 2. Jejunal Length and Weights of IUGR and NBW Piglets during the Suckling Perioda
Journal of Proteome Research • Vol. 9, No. 2, 2010
piglet
jejunal length (cm)
jejunal weight (g)
jejunal length index (cm/kg)b
jejunal weight index (%)c
IUGR 186.7 ( 5.3**d NBW 272.5 ( 4.5
Day 1 15.1 ( 0.33** 212.5 ( 11.2** 1.72 ( 0.03** 37.4 ( 0.87 187.0 ( 5.9 2.57 ( 0.12
IUGR 236.2 ( 16.3** NBW 346.2 ( 8.0
Day 7 48.3 ( 1.4** 121.8 ( 2.0 80.4 ( 2.1 131.1 ( 4.0
Day 21 IUGR 395.5 ( 21.2** 92.1 ( 4.4** NBW 594.2 ( 12.8 152.9 ( 3.2
77.1 ( 2.9** 91.1 ( 5.6
2.49 ( 0.07** 3.06 ( 0.09 1.79 ( 0.04** 2.35 ( 0.04
a Values are means ( SEM, n ) 6 per group. b Jejunal length index ) Jejunal length/Body weight. c Jejunal weight index ) Jejunal weight/Body weight × 100%. d **, P < 0.01 vs the NBW group.
Table 3. Jejunal Villus Heights and Widths of IUGR and NBW Piglets during the Suckling Perioda piglet
villus height (µm)
villus width (µm)
IUGR NBW
Day 1 329.8 ( 14.7**b 415.7 ( 17.2
26.4 ( 2.0* 36.5 ( 0.5
IUGR NBW
Day 7 353.7 ( 16.1** 476.3 ( 9.3
40.2 ( 1.7 37.1 ( 1.7
IUGR NBW
Day 21 215.3 ( 10.6** 386.3 ( 15.8
40.3 ( 1.8 41.2 ( 2.1
a Values are means ( SEM, n ) 6 per group. vs the NBW group.
b
*, P < 0.05; **, P < 0.01
than at D1. Jejunal villus width did not differ (P > 0.05) between IUGR and NBW piglets at D7 or D21. Representative morphologies of the jejunum from IUGR and NBW piglets are illustrated in Figure 1. At D1, scanning electron and light microscopic observations revealed thinner and atrophic villus tips in the jejunum of IUGR piglets (Figure 1, A1 and B1) compared with NBW (Figure 1, A2 and B2). Additionally, columnar epithelial cells and microvilli on the surface of jejunal villus in IUGR piglets were sparse, had anomalously loose array, and exhibited histological lesions (Figure 1, A1 and B1). In contrast, the jejunum of NBW piglets had more tight cell junctions and a normally oriented morphology (Figure 1, A2 and B2). Light microscopic examination showed the presence of edema beneath the layer of columnar epithelial cells in D7 IUGR jejunum (Figure 1, C1 and C2) and abnormally obscure appearance of jejunal brush border (microvilli) in D21 IUGR jejunum (Figure 1, D1 and D2). Temporal Analysis of Proteins. A total of 56 protein spots were differentially expressed in jejunum between IUGR and NBW piglets at D1, D7 and D21. Biochemical information about these protein spots is summarized in Tables 4 and 5, whereas their appearance on the gel images is labeled in Figure 2 and Figure 3. On the basis of their biological functions, these proteins are classified in nine groups: (1) absorption, digestion and transport; (2) cell structure and motility; (3) glucose and energy metabolism; (4) lipid metabolism; (5) amino acid metabolism; (6) mineral and vitamin metabolism; (7) cell redox homeostasis; (8) stress response; and (9) cellular apoptosis. Absorption, Digestion and Transport. Seven spots of proteins were related to absorption, digestion and transport of
Impairment of Intestinal Development in Neonatal Piglets
Figure 1. Representative histological pictures of scanning electron microscopy (A1, A2), as well as hematoxylin- and eosin-stained jejunum of IUGR (B1, C1, D1) and NBW (B2, C2, D2) piglets at Day 1 (B1 and B2), Day 7 (C1 and C2) and Day 21 (D1 and D2) of life.
nutrients. They are albumin (ALB, Spot L041, L161, L162, K081), chymodenin (Spot L2101), chymotrypsinogen B (CTRB, Spot L113), and clathrin light polypeptide isoform A (CLTA, Spot P020). Abundance of these proteins was consistently and continuously lower (P < 0.05) in the jejunal mucosa of IUGR piglets compared with NBW piglets at D1, D7 an D21. Cell Structure and Motility. Five spots of proteins play important roles in cell structure and motility. In comparison with NBW piglets, levels of ezrin (EZR, Spot K074), gammaactin (ACTG1, Spot L1368), and keratin 8 (KRT8, Spot K082) were lower (P < 0.05) in the jejunum of IUGR piglets at both D1 and D21, but higher (P < 0.05) at D7. In addition, COFILIN protein (CFL1, Spot K054) was down-regulated in the IUGR group at D1, D7 and D21. In contrast, abundance of Villin 1 (VIL1, Spot K073) was down-regulated in IUGR piglets at D7. Glucose and Energy Metabolism. Differentially expressed proteins that participate in energy metabolism include: enolase 1 (ENO1, Spot K084), fructose-bisphosphate aldolase A (FBPA, Spot K087), triosephosphate isomerase (TPI1, Spot L074), mitochondrial succinate dehydrogenase complex subunit A
research articles (SDHA, Spot L061), cytosolic glycerol-3-phosphate dehydrogenase (cGPDH, Spots L099, L1415), glycerol-3-phosphate dehydrogenase 1-like protein (GPD1L, Spot L075), cytochrome b5 (CYB5, Spot P027), cytochrome c oxidase subunit 5B, mitochondrial (COX5B, Spot P111), and creatine kinase (CK, Spots L1862, K099, K100). At D1, all of these proteins were reduced (>1.5-fold; P < 0.05) in the IUGR group. Among them, ENO1, FBPA, TPI1, cGPDH, GPD1L, CYB5, COX5B and CK were continuously down-regulated (P < 0.05) at D7 and/or D21, whereas cGPDH, GPD1L, CYB5, COX5B were up-regulated at D7. In addition, levels of SDHA were higher (P < 0.05) in the IUGR jejunum at both D7 and D21. Lipid Metabolism. Five spots of proteins are related to lipid metabolism. Abundance of apolipoprotein A-I (APOA1, Spot L094, P013) and apolipoprotein A-IV (APOA4, Spot L1146) was lower (P < 0.05) in the jejunal mucosa of IUGR piglets at D1, D7 and D21, when compared with NBW piglets. Additionally, at all 3 time-phases, levels of fatty acid binding protein 5 (FABP5, Spot P121) and fatty acid-binding protein 1 (FABP1, Spot L049) were lower (P < 0.05) at D1 and D21, but no difference was detected on D7, in comparison with NBW piglets. Amino Acid Metabolism. Four spots of jejunal proteins related to amino acid metabolism were affected by IUGR. Compared with NBW piglets, abundance of 3-hydroxyanthranilate 3,4-dioxygenase (3HAO, Spot K101), aminoacylase I (ACY1, Spot K085), and S-adenosylhomocysteine hydrolase (AHCY, Spot L006) was lower (P < 0.05) in the jejunum of IUGR piglets at D1 and D21, but was higher (P < 0.05) at D7. In contrast, levels of mitochondrial ornithine aminotransferase (OAT, Spot K076) were consistently reduced (P < 0.05) in the IUGR jejunum at all time points. Mineral and Vitamin Metabolism. Differentially expressed proteins related with mineral and vitamin metabolism include transferrin (TF, Spots K038, K039, K040, K089) and retinol binding protein 2, cellular (RBP2, Spots L137, L138). Levels of these proteins were lower (P < 0.05) in the jejunal mucosa of IUGR piglets than those in NBW piglets between D1 and D21. Abundance of another related protein, hemopexin (HPX, Spot P152), was down-regulated (P < 0.05) in the IUGR jejunal mucosa at both D7 and D21. Compared with NBW piglets, levels of haptoglobin (HP, Spot L189) in IUGR piglets were reduced (P < 0.05) at D1 and D21, but were increased (P < 0.05) at D7. Cell Redox Homeostasis. IUGR affected (P < 0.05) expression of a number of proteins involved in regulation of cell redox homeostasis. Specifically, abundance of beta-globin (BG, Spot K049) and protein disulfide isomerase-associated 3 (PDIA3, Spot P006) was reduced (P < 0.05) in the IUGR jejunal mucosa at D1 and D21, respectively, in comparison with NBW piglets. Levels of peroxiredoxin-1 (PRDX1, Spot K088), peroxiredoxin-5 (PRDX5, Spot P036) and chloride intracellular channel protein 1 (CLIC1, Spot K090) were lower (P < 0.05) in IUGR than in NBW piglets at D7. Interestingly, abundance of peroxiredoxin-6 (PRDX6, Spot L090) was reduced (P < 0.05) early at D1 in the IUGR jejunum compared with NBW piglets. Stress Response. Expression of several jejunal proteins to stress response was affected by IUGR. Of particular interest, heat shock 70 kDa protein 8/heat shock cognate 71 kDa protein (HSPA8/HSC70, Spot P153, Spot L181) were up-regulated (P < 0.05) in the IUGR group at D1, in comparison with NBW piglets. Abundance of 94 kDa glucose-regulated protein (GRP94, Spot K021) and glutathione S-transferase omega (GSTO, Spot P157) Journal of Proteome Research • Vol. 9, No. 2, 2010 927
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Journal of Proteome Research • Vol. 9, No. 2, 2010 gi|90200404
TPI1 APOA1 APOA1 APOA4 FABP5 FABP1
Lipid Metabolism L094 Apolipoprotein A-I [Sus scrofa] P013 Apolipoprotein A-I [Sus scrofa] L1146 Apolipoprotein A-IV [Sus scrofa] P121 Fatty acid binding protein 5 [Sus scrofa] L049 Fatty acid-binding protein [Sus scrofa]
Amino Acid Metabolism K101 3-hydroxyanthranilate 3,4-dioxygenase [Bos taurus]
214
gi|112980819
SDHA
3HAO
gi|115495835
gi|164359 gi|164359 gi|47523830 gi|89886167 gi|55742707
176
295 395 334 107 141
445
120 243
ENO1 FBPA 103
188
cGPDH gi|149714300
GPD1L gi|73990384
147
cGPDH gi|149714300
gi|87196501 gi|156120479
122 235 94 435 184
gi|125292 gi|194018722 gi|194018722 gi|353819 gi|55926217
Glucose and Energy Metabolism L1862 Creatine kinase [Canis familiariz] K100 Creatine kinase [Sus scrofa] K099 Creatine kinase [Sus scrofa] P027 Cytochrome b5 [Oryctolagus cuniculus] P111 Cytochrome c oxidase subunit 5B, mitochondrial [Sus scrofa] L1415 Cytosolic glycerol-3-phosphate dehydrogenase [Sus scrofa] L099 Cytosolic glycerol-3-phosphate dehydrogenase [Sus scrofa] K084 Enolase 1 [Bos taurus] K087 Fructose-bisphosphate aldolase A [Bos taurus] L075 Glycerol-3-phosphate dehydrogenase 1-like protein (GPD1L protein) [Bos taurus] L061 Mitochondrial succinate dehydrogenase complex subunit A [Sus scrofa] L074 Triosephosphate isomerase [Sus scrofa] CK CK CK CYB5 COX5B
EZR CFL1 ACTG1 KRT8 VIL1
Cell Structure and Motility K074 Ezrin [Bos taurus] (Villin-2) K054 COFILIN protein [Sus scrofa] L1368 Gamma-actin [Homo sapiens] K082 PREDICTED: similar to Keratin 8 [Sus scrofa] K073 PREDICTED: similar to Villin 1 [Sus scrofa] 238 202 105 701 156
140 92
gi|227130 gi|117616
CTRB gi|194033419 gi|51592135 gi|4501887 gi|227430407 gi|194043826
460 454 421 166 153
gi|833798 gi|833798 gi|833798 gi|124257959 gi|73953329
abbr.
protein scoreb
ALB ALB ALB ALB CLTA
protein name
accession no.
Absorption, Digestion and Transport Ability L162 Albumin [Sus scrofa] L161 Albumin [Sus scrofa] L041 Albumin [Sus scrofa] K081 Albumin [Sus scrofa] P020 PREDICTED: similar to clathrin, light polypeptide isoform A [Canis familiariz] L2101 Chymodenin [Sus scrofa domestica] L113 Chymotrypsinogen B [Bos taurus]
spot no.a
5.6 ( 0.5 8.4 ( 1.5 1.7 ( 0.9 2.8 ( 0.2 24.2 ( 4.8 4.6 ( 0.4 22.4 ( 1.2
1.1 ( 0.1 1.5 ( 0.2 1.2 ( 0.3 1.8 ( 0.3 0.9 ( 0.4 0.6 ( 0.3 4.3 ( 1.1
4.3 ( 0.7
10.1 ( 1.9
5.7 ( 0.5
4.2 ( 0.1
2.8 ( 0.1
6.6 ( 1.4
8.7 ( 0.2
22.1 ( 8.0 10.4 ( 1.6 1.8 ( 0.1 6.0 ( 0.8 87.8 ( 4.6
30.6 ( 6.0 21.3 ( 2.6
7.6 ( 1.2
32.2 ( 4.9 12.4 ( 3.1
10.5 ( 1.0 4.2 ( 0.3 ND 13.4 ( 0.6
9.1 ( 0.6
6.4 ( 0.1
ND 5.7 ( 0.4 ND 4.2 ( 0.8 ND 2.1 ( 0.6 16.2 ( 0.9 13.5 ( 0.8 32.9 ( 0.8 17.6 ( 1.0
1.5 ( 0.1 0.8 ( 0.1 16.9 ( 1.2 31.8 ( 9.0 4.3 ( 0.1 3.7 ( 0.8 25.8 ( 1.6 7.3 ( 1.0 6.3 ( 0.1 ND
2.2 ( 0.2 13.4 ( 2.5 21.2 ( 2.1 5.7 ( 1.9 13.8 ( 2.1 27.1 ( 9.0 ND 1.1 ( 0.3 2.9 ( 0.3 2.2 ( 0.6 8.1 ( 1.7 3.3 ( 0.1 14.6 ( 5.3 101.5 ( 31.2 234.2 ( 28.5
ND ND ND 15.3 ( 0.3 20.0 ( 1.4
2.1 ( 0.1 22.9 ( 4.4 2.6 ( 0.1 13.3 ( 0.2 1.0 ( 0.2
ND 8.3 ( 2.4 8.2 ( 0.7 17.0 ( 1.9
4.3 ( 0.7 2.6 ( 0.7
D1
9.3 ( 0.2 9.0 ( 2.8 12.0 ( 2.5 19.1 ( 7.3 96.1 ( 3.1 40.4 ( 13.7 5.2 ( 0.5 9.2 ( 0.4 8.4 ( 0.8 3.3 ( 1.1
D21
3.8 ( 0.6 7.9 ( 0.7 28.3 ( 11.3 1.7 ( 0.1 6.6 ( 0.8
D7
0.4 ( 0.1 ND ND 6.9 ( 2.4 5.3 ( 0.3
ND 15.5 ( 4.1 ND 3.6 ( 0.9 ND
ND 3.7 ( 0.2
NDd 1.0 ( 0.3 3.1 ( 0.3 2.7 ( 0.1 2.3 ( 0.1
D1
IUGR
expression level ( × 104)c
D21
42.2 ( 4.0
2.5 ( 0.3
40.2 ( 3.2
21.6 ( 1.1 ND
9.3 ( 0.3
8.6 ( 0.6
ND ND ND 27.2 ( 1.9 58.4 ( 0.7
2.7 ( 0.1 43.8 ( 0.5 6.0 ( 0.8 41.9 ( 0.5 6.1 ( 0.2
ND 19.5 ( 5.9
4.1 ( 1.1
14.1 ( 0.7
23.6 ( 3.0 41.0 ( 7.3 30.5 ( 6.9 46.6 ( 5/0 3.1 ( 0.6 6.1 ( 0.3 7.1 ( 0.7 16.5 ( 2.7 112.1 ( 26.1 246.6 ( 19.9
25.3 ( 2.4
2.4 ( 0.4
23.7 ( 3.0
8.1 ( 1.3 8.8 ( 0.5
8.0 ( 0.4
4.6 ( 0.6
ND 9.4 ( 0.2 6.9 ( 0.3 11.6 ( 2.7 12.7 ( 0.4
1.9 ( 0.2 58.5 ( 6.8 2.0 ( 0.2 8.9 ( 0.4 4.8 ( 0.4
16.1 ( 2.2 8.1 ( 1.0
13.9 ( 1.1 30.7 ( 3.1 33.7 ( 1.4 43.7 ( 3.1 93.4 ( 13.7 124.3 ( 34.4 4.1 ( 0.8 7.5 ( 0.1 8.5 ( 1.1 13.2 ( 1.9
D7
NBW
Table 4. Biochemical Information about Proteins Differentially Expressed in the Jejunal Mucosa of IUGR Piglets during the Suckling Period
