iTRAQ-based quantitative proteomic analysis of longissimus muscle from growing pigs with dietary supplementation of non-starch polysaccharide enzymes#

iTRAQ-based quantitative proteomic analysis of longissimus muscle from growing pigs with dietary supplementation of non-starch polysaccharide enzymes # # 基于iTRAQ定量蛋白质组学技术分析日粮中添加非淀粉多糖酶对生长猪背最长肌中蛋白表达影响 1 first-author 1 2 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhanghf6565@vip.sina.com †E-mail:zhanghf6565@vip.sina.com zhanghf6565@vip.sina.com State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China College of Animal Science and Technology, Jilin University, Changchun 130062, China College of Animal Science and Technology, Jilin University, Changchun 130062, China Non-starch polysaccharide enzymes (NSPEs) have long been used in the feed production of monogastric animals to degrade non-starch polysaccharide to oligosaccharides and promote growth performance. However, few studies have been conducted on the effect of such enzymes on skeletal muscle in monogastric animals. To elucidate the mechanism of the effect of NSPEs on skeletal muscle, an isobaric tag for relative and absolute quantification (iTRAQ) for differential proteomic quantitation was applied to investigate alterations in the proteome in the longissimus muscle (LM) of growing pigs after a 50-d period of supplementation with 0.6% NSPEs in the diet. A total of 51 proteins were found to be differentially expressed in the LM between a control group and the NSPE group. Functional analysis of the differentially expressed protein species showed an increased abundance of proteins related to energy production, protein synthesis, muscular differentiation, immunity, oxidation resistance and detoxification, and a decreased abundance of proteins related to inflammation in the LM of the pigs fed NSPEs. These findings have important implications for understanding the mechanisms whereby dietary supplementation with NSPEs enzymes can promote growth performance and improve muscular metabolism in growing pigs. 目的 2 通过日粮中添加非淀粉多糖酶，运用同位素标记相对和绝对定量技术（iTRAQ技术）分析非淀粉多糖酶对生长猪背最长肌蛋白质表达有何影响，为饲料中添加非淀粉多糖酶提供理论基础。 创新点 2 采用iTRAQ定量蛋白质组学技术，通过对生长猪背最长肌蛋白质表达进行高通量分析，发现日粮中添加非淀粉多糖酶可影响许多功能蛋白表达，从分子水平阐述了其发挥作用的机理。 方法 2 将体重约39kg生长猪（48头）随机分为两个处理，每个处理4个重复，每个重复6头猪。对照组饲喂基础日粮，试验组在基础日粮中添加0.6%非淀粉多糖酶。50天试验期后，每个重复屠宰1头猪（ n =8），取背最长肌，通过iTRAQ定量蛋白质组学技术分析肌肉组织中差异蛋白表达。 结论 2 iTRAQ定量蛋白质组学分析结果显示，试验组与对照组相比，共发现51个差异蛋白，其中38个可进行生物学功能定位（图1，表6）。差异表达蛋白中与能量生成、蛋白质合成、肌肉分化、免疫、抗氧化和解毒相关蛋白表达量上调，而与炎症反应相关蛋白表达量下调。综上所述，生长猪日粮中添加非淀粉多糖酶不仅可改善生产性能，同时还可调节肌肉中诸多代谢功能。 Non-starch polysaccharide enzymes (NSPEs) Longissimus muscle Proteomics Growing pigs 非淀粉多糖酶 背最长肌 蛋白质组学 生长猪 Introduction 1 Non-starch polysaccharide enzymes (NSPEs) are a group of exogenous enzyme mixtures, which have long been used in the feed production of monogastric animals to degrade non-starch polysaccharides (NSPs) to oligosaccharides (Silva and Smithard, 2002 2002 ; Bindelle et al., 2011 2011 ; Walsh et al., 2012 2012 ; Willamil et al., 2012 2012 ; Kiarie et al., 2013 2013 ). Researchers have proved that the addition of exogenous enzymes has multiple benefits by reducing the impact of numerous antinutritional factors in corn and soybean meal based diets (Yang et al., 2010 2010 ; Zou et al., 2013 2013 ). Previous studies have demonstrated that NSPEs can enhance animal growth performance and improve nutrient absorption and immunity, indicating that NSPEs play a versatile role in regulating metabolic pathways (Ao et al., 2010 2010 ; Zduńczyk et al., 2013 2013 ). Most studies have shown multiple benefits in the small intestine of pigs from adding NSPEs to the diet (Gdala et al., 1997 1997 ; Yin et al., 2001 2001 ; O'Shea et al., 2014 2014 ). However, only a few studies have been conducted on the effects of NSPEs on skeletal muscle in monogastric animals (Wang et al., 2005 2005 ; Buchanan et al., 2007 2007 ; Hajati et al., 2009 2009 ). Skeletal muscle is an important organ in the body, representing 40%–50% of body mass in mammals, and is a critical regulator that integrates various biochemical pathways, such as heat homeostasis and carbohydrate metabolism (Abdul-Ghani and DeFronzo, 2010 2010 ; Sandri, 2010 2010 ). Other specialized functions have also been found among the complex cellular tasks of muscles because of the actions of a number of different proteins (Ohlendieck, 2011 2011 ). Many of these are membrane-associated proteins including the nicotinic acetylcholine receptor (Fagerlund and Eriksson, 2009 2009 ), acetylcholinesterase (Aldunate et al., 2004 2004 ), the voltage-sensing dihydropyridine receptor (Arikkath and Campbell, 2003 2003 ), the ryanodine receptor Ca 2+ -release channel (Franzini-Armstrong, 2009 2009 ), the dystrophin-glycoprotein complex (Michele and Campbell, 2003 2003 ), and the respiratory chain (Hood, 2009 2009 ). However, little is known about the exact molecular mechanisms of the action of NSPEs on skeletal muscle (Hajati et al., 2009 2009 ). Numerous studies have demonstrated a lack of correlation between mRNA and protein abundance because of RNA editing and post-translational modifications (Ohlendieck, 2011 2011 ). Making the elucidation of protein expression more accurate is imperative (Wang et al., 2006 2006 ). Recent research has applied advanced high throughput mass spectrometric proteomic technologies to the simultaneous measurement of the expression of hundreds of proteins (Kitteringham et al., 2010 2010 ; Luo et al., 2013 2013 ). A proteomic analysis of skeletal muscle in rainbow trout demonstrated that previously unrecognized proteins were involved in various functions, including energy production, protein biosynthesis and modification, inflammation, the immune response, and transcriptional regulation (Salem et al., 2010 2010 ). There is great interest in using proteomic technology to elucidate the beneficial effects brought about by NSPEs in the skeletal muscle of growing pigs. Due to the large number of membrane-associated proteins and some highly-abundant proteins in the skeletal muscle, results from two-dimensional electrophoresis may be misleading. Therefore, in this study, we used a label-based isobaric tag for relative and absolute quantification (iTRAQ) procedure, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitate the altered proteins that are induced differentially in the longissimus muscle (LM) of growing pigs fed NSPEs. Materials and methods 1 Enzyme preparation 2 The NSP enzyme mixture used in the present study was supplied by the State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (Beijing, China), and contains 7×10 5 U/g xylanase (EC 3.2.1.8), 1×10 5 U/g β-glucanase (EC 3.2.1.6) and 9000 U/g cellulase (EC 3.2.1.4). The activities of xylanase, β-glucanase, and cellulase were measured according the methods of Bailey and Poutanen ( 1989 1989 ), Erfle et al. ( 1988 1988 ), and Lowe et al. ( 1987 1987 ), respectively. Reagents and chemicals 2 A Protein Assay Kit was purchased from Bio-Rad (Hercules, CA, USA). Reagent for total RNA isolation was obtained from Qiagen (Valencia, CA, USA). Reagents for quantitative polymerase chain reaction (qPCR) were obtained from TaKaRa Biotechnology (Dalian, China). All iTRAQ reagents and buffers were obtained from Applied Biosystems Inc. (ABI, Foster City, CA, USA). Other reagent grade chemicals used in the study were obtained from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburg, PA, USA). Animals and treatments 2 Forty-eight crossbred (Duroc×Landrace×Large White) growing pigs with similar initial body weights ((39.18±0.98) kg) were purchased from a local pig farm in Beijing. Pigs were randomly assigned to 2 groups according to their littermates and mean initial body weights, with 4 replicates in each group and 6 pigs in each replicate (3 males and 3 females). The 2 groups comprised a control group (CTRL) (fed basal diet) and a treatment group (NSPE) (fed basal diet supplemented with 0.6% NSPEs). Both diets were formulated to meet National Research Council (NRC, 2012 2012 ) recommendations (Table 1 1 ). The animals were housed in a fermentation bed facility in 8 adjacent pens. Feed and water were provided ad libitum during the 50-d experimental period. The weight and feed intake of each pig were recorded at the beginning and the end of the experiment for the calculation of average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). All procedures involving animals were evaluated and approved by the Animal Ethics Committee of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences. All data are expressed in g/kg dry weight except for digestible energy in MJ/kg Providing the following (g/kg fresh weight): vitamin A 8250 IU, vitamin D 3 825 IU, vitamin E 40 IU, vitamin K 3 4.0 mg, vitamin B 1 1.0 mg, vitamin B 2 5.0 mg, vitamin B 6 2.0 mg, vitamin B 12 25 μg, choline chloride 600 mg, nicotinic acid 35 mg, folic acid 2.0 mg, biotin 4.0 mg, Cu 50.0 mg, Fe 80.0 mg, Zn 100.0 mg, Mn 25.0 mg, Se 0.15 mg, iodine 0.5 mg Composition Proportion (%) * * Ingredients  Corn 70.70  Soybean meal 19.82  Soybean oil 2.10  Wheat bran 5.00  Limestone 0.51  Calcium hydrophosphate 0.56   L -Lysine 0.01  Sodium chloride 0.30  Premix a a 1.00  Total 100 Nutrient  Digestible energy 14.22 (MJ/kg)  Ether extract 4.82  Crude protein 15.50  Calcium 0.50  Total phosphorus 0.45  Total lysine 0.75  Total methionine 0.25  Available phosphorus 0.24 Sample collection and meat quality analysis 2 At Day 50 (a total 50 d of feeding treatment), all pigs were weighed after 12 h of fasting. One pig per replicate ( n =8) was sacrificed by electrical stunning, and then exsanguinated. Blood samples were collected from the cervical vein before sacrifice. After collection, blood samples were centrifuged at 2000 g for 30 min at 4 °C, then at 400 g for 10 min at 4 °C. The sera obtained were stored at −20 °C for further analysis. The whole LM was obtained from the right side of the carcass, as previously described (Zhong et al., 2011 2011 ). LM samples collected for proteomic analysis were snap-frozen in liquid nitrogen and stored at −80 °C. The loin-eye area was measured by the method described by Turyk et al. ( 2014 2014 ). The meat quality of the LM was analyzed as previously described (Liu et al., 2014 2014 ), including the pH values (after 45 min and 24 h), drip loss, and shear force. The chemical composition of the LM, including its intramuscular fat, moisture, protein, and ash content, was measured as described by Huang et al. ( 2014 2014 ). Serum biochemical analyses 2 For biochemical analysis, serum levels of total cholesterol, triglyceride, low density lipoprotein-cholesterol (LDL-C), and high density lipoprotein-cholesterol (HDL-C) were analyzed using a corresponding kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions of the manufacturer. The blood urea nitrogen (BUN) was measured by the Abbott Spectrum urea nitrogen test (Series II, Abbot Laboratories, Dallas, TX, USA). Protein extraction 2 Protein extraction was performed as previously described with some modifications (Shao et al., 2010 2010 ; Ramadoss and Magness, 2012 2012 ). LM samples (500 μg) were ground in a Dounce glass grinder using liquid nitrogen. Ground samples were precipitated with 10% (0.1 g/ml) trichloroacetic acid (TCA), in 90% ice-cold acetone at −20 °C for 2 h. The samples were then centrifuged at 20 000 g for 30 min at 4 °C. The supernatants were decanted and the pellets washed with ice-cold acetone. The pellets were lysed in lysis buffer consisting of 8 mol/L urea, 30 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), 2 mmol/L ethylene diamine tetraacetic acid (EDTA), and 10 mmol/L dithiothreitol (DTT). The undissolved pellets in the crude tissue extracts were removed by centrifugation (20 000 g , 30 min, 4 °C). The tissue lysates were reduced with 10 mmol/L DTT for 1 h at 36 °C, and then alkylated with 55 mmol/L iodoacetamide (IAM) for 1 h at room temperature in the dark. After reduction and alkylation, 4 volumes of ice-cold acetone were added to the solution to precipitate proteins. The proteins were washed three times with ice-cold pure acetone and re-dissolved in buffer containing 50% tetraethyl ammonium bromide (TEAB) and 0.1% sodium dodecyl sulfonate (SDS). The undissolved pellets were removed from protein samples by centrifugation (20 000 g , 30 min, 4 °C), and protein quantitation was carried out using a Bio-Rad Bradford Protein Assay Kit (Hercules, CA, USA). Trypsin digestion 2 Each protein sample was digested overnight at 37 °C by adding sequencing grade trypsin (Promega Co., Madison, WI, USA) at a 1:30 ratio (3.3 μg trypsin: 100 μg target) (Ramadoss and Magness, 2012 2012 ). iTRAQ labeling 2 The iTRAQ labeling procedure was described by Ramadoss and Magness ( 2012 2012 ). All protein samples were labeled with iTRAQ tags (solubilized in 70 μl isopropanol). Control samples received tags 113, 114, 115, and 116, while treatment samples received tags 117, 118, 119, and 121. All labeled samples (organic composition > 60% by adding isopropanol) were incubated at room temperature for 2 h. Strong cation exchange chromatography 2 The strong cation exchange fractionation protocol followed previous reports (Olsen et al., 2006 2006 ; Shao et al., 2010 2010 ; Su et al., 2013 2013 ) with slight modifications. Briefly, a total amount of 800 μg sample was loaded onto a strong cation exchange column (Phenomenex Luna SCX 100A) equilibrated with buffer A (10 mmol/L KH 2 PO 4 in 25% acetonitrile, pH 3.0) using an Agilent 1100 system (Santa Clara, CA, USA). The peptides were separated using a linear gradient of buffer B (10 mmol/L KH 2 PO 4 and 2 mol/L KCl in 25% acetonitrile, pH 3.0) increasing to 5% after 41 min, 50% after 66 min, and 100% after 71 min, at a flow rate of 1 ml/min. A total of 10 fractions were collected from the eluted peptides, and each fraction was desalted with a Strata X C18 column (Phenomenex, Torrance, CA, USA) and vacuum-dried using a Speedvac (Thermo Fisher Scientific, Bremen, Germany). Mass spectrometry 2 The protocol for MS analysis was as described previously (Su et al., 2013 2013 ) with slight modifications. Each SCX fraction was re-dissolved in buffer A (2% acetonitrile, 0.1% formic acid) and centrifuged at 20 000 g for 10 min. The final concentration of peptides in each fraction was about 0.25 μg/μl on average. Twenty microliters of supernatants were loaded by an auto sampler onto a C18 trap column (length 2 cm, inner diameter 200 μm) of an UltiMate® 3000 Nano LC system (Bannockburn, IL, USA). Peptides were eluted onto a resolving analytical C18 column (length 10 cm, inner diameter 75 μm, 5 μm particles, 30 nm) packed in-house. Samples were loaded at 15 μl/min for 4 min and eluted with a 45 min gradient at 400 nl/min from 5% to 60% buffer B (98% acetonitrile, 0.1% formic acid), separated with a 3-min linear gradient to 80% B, maintained at 80% B for 7 min, and finally returned to 5% B over 3 min. The peptides were subjected to nanoelectrospray ionization followed by tandem mass spectrometry (Q-Exactive, Thermo) coupled online to the nanoLC. Intact peptides were detected in the Orbitrap at a resolution of 70 000 full width at half maximum (FWHM). Peptides were selected for MS/MS using the high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 28%; ion fragments were detected in the Orbitrap at 17 500 FWHM resolution. A data-dependent acquisition mode that alternated between an MS scan and MS/MS scans was applied for the 10 most abundant precursor ions (2 + to 4 + ) above a threshold ion count of 20 000 in the MS survey scan, with a following dynamic exclusion duration of 15 s (isolation window of m/z 2.0 and a maximum ion injection time of 100 ms). The electrospray voltage applied was 1.8 kV. Automatic gain control (AGC) was used to optimize the spectra generated by the Orbitrap. The AGC target for full MS was 3e6 and 1e5 for MS2. For MS scans, the m/z scan range was 350–2000. For MS2 scans, the m/z scan range was 100–1800. Data processing and analyses 2 MS/MS data for iTRAQ protein identification and quantitation were analyzed using Proteome Discover 1.3 (Thermo Fisher Scientific, Bremen, Germany) and searched using in-house MASCOT software (Matrix Science, London, UK; Version 2.3.0) against the database Uniprot_pig (Apr. 11th, 2014) with the following parameters: enzyme: trypsin; fixed modification: carbamidomethyl (C); variable modifications: oxidation (M), gln-pyro-glu (N-term Q), iTRAQ 8-plex (N-term, K, Y); peptide mass tolerance:15 ppm; MS/MS tolerance: 20 mmu; maximum missed cleavages: 1. Identified peptides had an ion score above the threshold of peptide identity established by Mascot, and protein identifications were accepted within the false discovery rate (FDR) of 1% in which at least one such unique peptide match was specific for the protein. Median ratio normalization was performed in intra-sample channels to normalize each channel across all proteins. Protein quantitative ratios for each iTRAQ labeled sample were obtained, using a sample in the control group (sample tagged with 113) as the denominator. Quantitative ratios were then log transformed to base 2 and presented as fold change relative to the denominator in the control group for final quantitative testing. Differentially expressed proteins were identified using Student’s t -test corrected for multiple testing using the Benjamini and Hochberg correction (Hakimov et al., 2009 2009 ). Proteins with a 1.2-fold change or greater were considered to be differentially expressed. Bioinformatics analysis of protein differential abundance 2 Gene Ontology (GO) distributions for all of the proteins that were significantly altered in the LM in growing pigs fed NSPEs were classified using Blast2GO ( http://www.blast2go.com http://www.blast2go.com ) and WEGO ( http://wego.genomics.org.cn http://wego.genomics.org.cn ) software provided by the Institute for Genomic Research (Ye et al., 2006 2006 ; Zi et al., 2013 2013 ). Validation of proteins of differential abundance 2 Real-time qPCR was used to verify six LM proteins of differential abundance at the mRNA level by the method of Huang et al. ( 2013 2013 ) with slight modifications. Total RNA from the LM was isolated using a Qiagen RNeasy Plus Mini Kit (Valencia, CA, USA). Agarose gel electrophoresis was used to test the RNA quality. The RNA quantity was verified with a spectrophotometer (Nanodrop 2000, Thermo Scientific, Waltham, MA, USA). Then total RNA was reverse transcribed using the PrimeScrip™ Reverse Transcriptase, D2680A (TaKaRa Biotechnology, Dalian, China). All mRNA expression levels were analyzed by RT-qPCR using the PrimeScript™ RT reagent Kit, RR037A (SYBR Green) (TaKaRa Biotechnology, Dalian, China) and an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). RT-qPCR was performed under the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Fluorescence data were detected at the last step of each cycle to monitor the amount of PCR product. The primer sequences are shown in Table S1 S1 . The relative fold-change was calculated by the 2 −ΔΔ C T method (Huang et al., 2013 2013 ). Statistical analysis 2 Data on growth performance, serum parameters, and gene expression were analyzed by one-way analysis of variance (ANOVA; SAS Version 9.2, SAS institute Inc., Cary, NC, USA). A group difference was assumed to be statistically significant when P < 0.05. All results are expressed as means±standard deviation (SD). Results 1 Growth performance of growing pigs 2 In this study, all growing pigs had similar body weights ((39.18±0.98) kg) at the initiation of the experiment. During the entire experimental period (total 50 d), NSPE pigs increased their body weight by 5.9% and their ADG by 15.5% ( P < 0.05). However, the ADFI between the two groups was similar ( P > 0.05). Supplementation with NSPEs significantly improved FCR by 8.7% compared with the control group ( P < 0.05; Table 2 2 ). CTRL: control; NSPE: non-starch polysaccharide enzyme; ADG: average daily gain; ADFI: average daily feed intake; FCR: feed conversion ratio. Values are presented as mean±SD ( n =24 for ADG; n =4 for ADFI and FCR). Values within a column having different superscript letters indicate a significant difference at P < 0.05 Group Initial weight (kg) Final weight (kg) ADG (kg/d) ADFI (kg/d) FCR (kg feed/kg weight gain) CTRL 38.80±0.99 74.04±1.77 b 0.71±0.05 b 1.97±0.09 2.77±0.02 a NSPE 39.55±0.63 78.42±1.06 a 0.82±0.05 a 2.07±0.06 2.53±0.03 b Meat quality and composition of growing pigs 2 Most indexes of carcass characteristics, meat quality and meat chemical composition of pigs fed NSPEs were not different ( P > 0.05) from those of pigs fed the CTRL diet, except for loin-eye area, which was significantly greater in the NSPE group ( P < 0.05) (Tables 3 3 and 4 4 ). CTRL: control; NSPE: non-starch polysaccharide enzyme. Values within a column having different superscript letters indicate a significant difference at P < 0.05. Values are presented as mean±SD ( n =4) Group Carcass length (cm) Skin thickness (cm) Average backfat (cm) Loin-eye area (cm 2 ) LM length (cm) CTRL 101.7±5.0 0.92±0.16 2.46±0.57 41.85±3.14 b 82.00±1.87 NSPE 102.3±5.7 0.93±0.16 2.56±0.47 45.97±4.29 a 81.78±1.20 CTRL: control; NSPE: non-starch polysaccharide enzyme. Values are presented as mean±SD ( n =4) 2 Group 4 Meat quality traits 4 Chemical composition pH 45 min pH 24 h Drip loss (%) Shear force (kg) Moisture (%) Intramuscular fat (%) Crude protein (%) Ash (%) CTRL 5.66±0.21 5.45±0.08 9.31±2.30 3.57±0.17 75.50±1.71 1.36±0.09 24.80±1.00 1.11±0.12 NSPE 5.66±0.20 5.54±0.06 9.12±2.01 3.12±0.22 75.70±2.10 1.42±0.11 24.40±1.30 1.12±0.13 Serum parameters of growing pigs 2 Among the serum parameters, only the level of LDL-cholesterol was significantly lower in the NSPE group than in the CTRL group (Table 5 5 ; P < 0.05). CTRL: control; NSPE: non-starch polysaccharide enzymes; BUN: blood urea nitrogen; LDL-C: low density lipoprotein-cholesterol; HDL-C: high density lipoprotein-cholesterol. Values within a column having different superscript letters indicate a significant difference at P < 0.05. Values are presented as mean±SD ( n =4) Group BUN (mmol/L) Triglyceride (mmol/L) Cholesterol (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) CTRL 7.09±1.69 0.62±0.23 2.66±0.42 1.60±0.25 a 1.09±0.12 NSPE 7.07±1.23 0.52±0.08 2.31±0.41 1.22±0.19 b 1.00±0.23 Identification and comparison of proteins of differential abundance 2 Using iTRAQ analysis, a total of 1167 proteins were identified within the FDR of 1% (supporting information, Table S2 S2 ). Following statistical analysis, 51 proteins were found to be differentially expressed in the LM between CTRL and NSPE pigs, with 21 being up-regulated and 30 down-regulated (supporting information, Table S3 S3 ). A total of 38 proteins of differential abundance were grouped into seven classes based on putative functions: protein degradation, biosynthesis and modification (26.3%), transcriptional and translational regulation (18.4%), inflammatory and immune response (15.8%), miscellaneous (13.2%), lipid metabolism (10.5%), adenosine triphosphate (ATP) production and substrate metabolism (7.9%), and oxidative stress and detoxification (7.9%) (Fig. 1 1 ). Those related to protein degradation, biosynthesis and modification, transcriptional and translational regulation, and inflammatory and immune response were predominant, accounting for about 60% of the differentially-expressed proteins. A comparison of proteins of differential abundance with functional groupings between the two groups indicated that a smaller number of protein species were up-regulated in NSPE pigs (15 versus 23) (Table 6 6 ). CTRL: control; NSPE: non-starch polysaccharide enzyme. NSPE supplementation significantly altered 51 proteins with 21 being up-regulated and 30 down-regulated. A total of 38 proteins of differential abundance were grouped into seven classes based on putative functions. Protein expression is represented as log 2 fold relative to the control Accession Description Gene symbol log 2 fold change P -value Biological process GO term 2 ATP production and substrate metabolism  O19069 Succinyl-CoA ligase [ADP/GDP-forming] subunit α SUCLG1 −0.14 0.0194 GTP binding  F1SDJ8 Uncharacterized protein GLRX3 0.08 0.0382 Electron carrier activity  P11708 Malate dehydrogenase, cytoplasmic MDH1 0.12 0.0132 Tricarboxylic acid cycle 2 Lipid metabolism  F1SJT7 Apolipoprotein A-IV APOA4 −0.07 0.0307 Cholesterol transporter activity  M9TGS8 Mitochondrial δ3,δ2-dienoyl-CoA isomerase ECI1 0.17 0.0282 Fatty acid β-oxidation  P49924 Fatty acid-binding protein FABP1 1.03 0.0045 Lipid binding  F1STB2 Uncharacterized protein HINT2 0.19 0.0407 Mitochondrial lipid metabolism and respiration 2 Transcriptional and translational regulation  Q53DY6 Histone H1.3-like protein −1.05 0.0051 DNA binding  B6V8C8 40S ribosomal protein S3a RPS3A −0.30 0.007 Structural constituent of ribosome  A1XQU3 60S ribosomal protein L14 RPL14 −0.26 0.0341 Structural constituent of ribosome  I3LVI8 60S ribosomal protein L7a RPL7A −0.24 0.0446 Ribosome biogenesis  Q29293 60S ribosomal protein L3 RPL3 0.36 0.0253 Structural constituent of ribosome  I3LKZ4 Uncharacterized protein HNRNPR −0.26 0.0308 Nucleotide binding  I3LIL4 Uncharacterized protein MYO1C −0.44 0.0345 Motor activity 2 Protein degradation, biosynthesis and modification  K7GKE7 Haptoglobin −1.03 0.0184 Proteolysis  I6LNT8 Glycogen synthase kinase 3 α GSK3A −0.31 0.0158 Protein serine/threonine kinase activity  G9F6X9 Dihydropyrimidinase-like 2 −0.29 0.0177 Hydrolase activity  I3LD20 Dystroglycan DAG1 −0.38 0.0055 Structural constituent of muscle  P37111 Aminoacylase-1 ACY1 0.15 0.0007 Cellular amino acid metabolic process  Q29549 Clusterin CLU −0.26 0.0128 Ubiquitin protein ligase binding  D0G0C8 Chaperonin containing TCP1, subunit 2 (β) CCT2 −0.19 0.039 Actin-binding  I3LUK9 Ubiquitin-conjugating enzyme E2 D2 UBE2D2 −0.15 0.0334 Ubiquitin-protein ligase activity  F1SID4 Uncharacterized protein PSMC3 −0.15 0.0325 Ubiquitin-dependent process  D0G0C9 Chaperonin containing TCP1, subunit 7 CCT7 −0.14 0.0303 Protein folding 2 Inflammatory and immune response  K9J6K1 Tapasin TAPBP −0.59 0.0228 Antigen processing  F1SH92 Inter-α-trypsin inhibitor heavy chain H4 ITIH4 −0.47 0.0018 Acute-phase response  F1SHR2 Uncharacterized protein BCL2L13 −0.26 0.0002 Regulation of apoptotic process  Q29014 α-1 acid glycoprotein AGP −0.19 0.0454 Acute-phase response  L8B180 IgG heavy chain IGHG 0.51 0.0469 Immune response  K7GQ48 Uncharacterized protein A2M 0.88 0.0393 Complement and coagulation cascades 2 Oxidative stress and detoxification  I3LEF2 Uncharacterized protein NXN −0.46 0.0275 Thioredoxin-disulfide reductase activity  Q9N1F5 Glutathione S -transferase ω-1 GSTO1 0.09 0.0066 Metabolism of xenobiotics by cytochrome P450  F1SR94 Uncharacterized protein 0.4 0.0319 Metabolism of xenobiotics by cytochrome P450 2 Miscellaneous  I3LUN7 Uncharacterized protein KCNS3 −3.50 0.0184 Voltage-gated potassium channel activity  I3LJ91 Uncharacterized protein FHOD1 0.13 0.0251 Actin filament organization  F1SRI8 Uncharacterized protein MYBPC1 0.13 0.042 Muscle contraction  C1L369 Parvalbumin PVALB1 0.51 0.0274 Calcium ion binding  B3STX9 Prothrombin 0.19 0.0127 Calcium ion binding GO annotations and functional analysis 2 In the cellular component group, the differentially expressed proteins were concentrated in intracellular organelles and the cytoplasm (Fig. 2 2 ). In the molecular functional group, the differentially expressed proteins that are metabolic enzymes (hydrolase, oxidoreductase, or ligase activity) and binding proteins (protein, ion, or nucleoside binding) were ranked at the top of the category, suggesting that the relevant functions were important in the LM of pigs (Fig. 2 2 ). In the biological process category, the proteinsthat participate in cellular processes (protein metabolic process), metabolism (nitrogen compound metabolic process), and biological regulation (signal transduction), had the highest ratios among the differentially expressed proteins (Fig. 2 2 ), suggesting that supplementation with NSPEs results mainly in changes in the protein metabolism in the LM of pigs. The number of proteins for each GO annotation is shown on the right axis, and the proportion of proteins for each GO annotation is shown on the left axis Validation of proteins of differential abundance 2 Six differentially expressed proteins (aminoacylase-1 (ACY1) involved in protein biosynthesis, prothrombin involved in the differentiation of skeletal muscle, malate dehydrogenase (MDH1) involved in ATP production, 40S ribosomal protein S3a (RPS3A) involved in transcriptional and translational regulation, chaperonin containing TCP1, subunit 2 (β) (CCT2) involved in protein modification and immunoglobulin heavy chain γ polypeptide (IGHG) involved in immune response) were selected for validation of proteomic data at the mRNA level using qPCR (Fig. 3 3 ). Most protein levels were consistent with their mRNA expression levels, except for CCT2. Samples were normalized with the reference gene β-actin . Data are presented as mean±SD ( n =4). Mean values with different letters are significantly different ( P < 0.05) Discussion 1 The value of providing NSPEs to promote growth performance has been reported in many studies (Bindelle et al., 2011 2011 ; Willamil et al., 2012 2012 ; Kiarie et al., 2013 2013 ). However, the effects of NSPEs on meat quality and chemical composition were not obvious in the present study, which is consistent with previous studies (Kim et al., 2011 2011 ; Świątkiewicz et al., 2013 2013 ). As an important organ, skeletal muscle plays vital roles in multiple metabolic pathways, such as energy metabolism, which are highly relevant to growth (Suryawan and Davis, 2014 2014 ). Hence, it is possible that the alteration of metabolism in the skeletal muscle is related to supplementation with NSPEs in the diet. However, the underlying mechanisms are largely unknown. In the current study, we observed that several specific pathways were altered in the LM of growing pigs via supplementation with NSPEs, including energy metabolism, protein degradation, biosynthesis and modification, inflammation and immune response, oxidative stress and detoxification, and some other functions. The proteomic data obtained from the present study were also verified by mRNA expression levels. Most of the protein level were consistent with their corresponding mRNA expression levels, except for CCT2. Possible reasons for the inconsistent result may be the existence of RNA editing and post-translational modifications (Ohlendieck, 2011 2011 ; Luo et al., 2013 2013 ). Energy is a major resource needed for maintaining the normal physiological function of skeletal muscle, and is critical for muscle growth, development, and contraction (Gordon et al., 2000 2000 ; Ohlendieck, 2011 2011 ). In some pathological or stress conditions, for instance diabetes and fasting, the expression of proteins related to glucose use and the production of ATP is reduced, which triggers atrophy of skeletal muscle (Lecker et al., 2004 2004 ). In the present study performed in vivo with pigs fed NSPEs, we found a greater abundance of proteins, such as glutaredoxin 3 (GLRX3) and MDH1, that are involved in ATP production. GLRX3 may serve as an electron carrier in the respiratory chain for ATP synthesis (Cha and Kim, 2009 2009 ). MDH1 is an important enzyme in the tricarboxylic acid cycle that may provide H + for producing ATP in the next step (Minárik et al., 2002 2002 ). Increased abundance of GLRX3 and MDH1 in NSPE supplemented pigs could contribute to enhanced production of ATP in the LM. In contrast, succinyl-CoA ligase [ADP/GDP-forming] subunit α (SUCLG1), which serves as a GTP-binding protein in the formation of metabolic intermediates, was down-regulated in the treatment group. This requires additional research (Ottaway et al., 1981 1981 ). Protein species related to lipid metabolism were also up-regulated in pigs fed NSPEs. Of these proteins, mitochondrial δ3,δ2-dienoyl-CoA isomerase (ECI1) acts as an auxiliary enzyme in the β-oxidation of polyunsaturated fatty acids (Gurvitz et al., 1998 1998 ). Histidine triad nucleotide binding protein 2 (HINT2) is positively responsible for regulating mitochondrial lipid metabolism and respiration in the livers of mice (Martin et al., 2013 2013 ). Fatty acid-binding protein 1 (FABP1) facilitates the transportation of lipids in the body (Furuhashi and Hotamisligil, 2008 2008 ). Increased expression of these proteins may enhance the catabolism of lipids in the LM to stimulate the production of energy for growth. Apolipoprotein A-IV is associated with the secretion of triglyceride in the liver (VerHague et al., 2013 2013 ). Down-regulation of this protein was observed in the LM of NSPE-supplemented pigs, whereas the serum level of triglyceride was not different between the two groups. This behavior is consistent with previous research which showed that supplementation with NSPEs had no effect on serum level of total triglyceride (Kim et al., 2013 2013 ). Interestingly, down-regulated expression of proteins related to transcriptional and translational regulation, such as histone (histone H1.3-like protein) and ribosomal proteins (RPS3A, 60S ribosomal protein L14 and 60S ribosomal protein L7a), was observed in the LM of pigs fed NSPEs (Ramadoss and Magness, 2012 2012 ). Salem et al. ( 2010 2010 ) demonstrated that protein synthesis is reduced during stress or pathological conditions, while expression of proteins related to transcriptional and translational regulation is elevated. Based on the results of the current study, we speculate that supplementation with NSPEs in the diet of growing pigs may reduce the possibility of muscle atrophy. Furthermore, the lower abundance of haptoglobin, glycogen synthase kinase 3 α (GSK3A), and dihydropyrimidinase-like 2 observed in the LM of pigs fed NSPEs may further protect proteins from proteolysis under stress (Wassler and Fries, 1993 1993 ; Woodgett, 1994 1994 ; Wang and Strittmatter, 1997 1997 ). Protein species that participate in the modification of protein, including clusterin (CLU), CCT2, ubiquitin-conjugating enzyme E2 D2 (UBE2D2), PSMC3 (proteasome (Prosome, Macropain) 26S subunit, ATPase, 3), and CCT7 (chaperonin containing TCP1, subunit 7), were down-regulated in the treatment group (Lecker et al., 2004 2004 ). This pattern is in concert with expression of proteins related to transcriptional and translational regulation, and indicates that the two clusters of protein in the treatment group may have a similar function in preventing muscle atrophy. The growth of the LM depends mainly on protein synthesis. ACY1 plays an important role in protein synthesis by increasing the availability of amino acids in the serum (Welberry Smith et al., 2013 2013 ). Up-regulated expression of this protein was observed in the LM of growing pigs supplemented with NSPEs, which may explain why the loin-eye area was greater in pigs fed NSPEs compared with those in the control group. This outcome is consistent with previous research which showed that supplementation with NSPEs in the diet improved the loin-eye area in growing pigs (Uthai et al., 2004 2004 ). IGHG, an important antibody subunit, is composed of an antibody with an immunoglobulin light chain, and can be produced with the initiation of an immune response (Janeway et al., 2001 2001 ). α-2-Macroglobulin (A2M) may be involved in the pathway of complementation and coagulation cascades to eliminate pathogens (Turnberg and Botto, 2003 2003 ). The abundance of these two proteins was elevated in the LM of pigs fed NSPEs. This indicates that supplementation with NSPEs could enhance immunity in animals. In contrast, as a transporter associated with antigen processing, the abundance of tapasin was lower in the LM of pigs supplemented with NSPEs. This behavior is consistent with previous research showing the overexpression of tapasin in muscle atrophy (Salem et al., 2010 2010 ). Moreover, inter-α-trypsin inhibitor heavy chain H4 (ITIH4) and α-1 acid glycoprotein (AGP) are acute-phase proteins (APP), which are induced by inflammation-related cytokines (Fournier et al., 2000 2000 ; Piñeiro et al., 2004 2004 ). Also, BCL2-like 13 (apoptosis facilitator) (BCL2L13) is a member of the Bcl-2 family of proteins, and its overexpression can induce apoptosis (Kataoka et al., 2001 2001 ). Reduced abundance of these proteins could attenuate inflammation in the LM of the NSPE group. Along with the impact of immunity and inflammation, we also noticed that supplementation with NSPE had effects on the expression of proteins related to the stress response and detoxification. Nucleoredoxin (NXN) can regulate the formation of reactive oxygen species (ROS) (Hirota et al., 2000 2000 ; Funato and Miki, 2007 2007 ). Reduced expression of NXN in NSPE pigs attenuated oxidative stress in the LM. Glutathione S -transferase ω 1 (GSTO1) plays an important role in the detoxification of different drugs, carcinogens, and endogenous compounds by catalyzing the conjugation of glutathione with electrophilic substrates (Mukherjee et al., 2006 2006 ). Thus, a higher abundance of GSTO1 in NSPE pigs may facilitate the elimination of toxins in the LM. Notably, increased abundance of two calcium binding proteins, parvalbumin and prothrombin, was observed in the treatment group in our study. Parvalbumin is found in fast-twitch fibers and binds cytosolic Ca 2+ , enabling rapid relaxation after contraction (Ottaway et al., 1981 1981 ). Prothrombin is highly expressed by skeletal and vascular smooth muscle (McBane et al., 1997 1997 ), and its expression is up-regulated in murine skeletal muscle cells in response to differentiation (Kim and Nelson, 1998 1998 ). Combined with the results of the current study, we assume that elevated expression of parvalbumin and prothrombin in NSPE-supplemented pigs may help the skeletal muscle maintain normal physiological function and stimulate differentiation. Conclusions 1 In summary, the results of this study provide the first evidence for an altered abundance of proteome in the LM of growing pigs supplemented with NSPEs. Though the meat quality and chemical composition of the LM were not affected by NSPEs, protein species related to protein metabolism were significantly changed, which may be associated with the growth of skeletal muscle. Furthermore, supplementation with NSPEs resulted in additional benefits by facilitating an enhanced immune response, oxidation resistance and detoxification in the LM of pigs. In contrast, protein species relevant to inflammation were down-regulated in pigs fed NSPEs. 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Effects of exogenous enzymes and dietary energy on performance and digestive physiology of broilers The authors are grateful for the suggestions during drafting and revising manuscript by Dr. Qing-shi MENG (State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China). Compliance with ethics guidelines Ji-ze ZHANG, Yang GAO, Qing-ping LU, Ren-na SA, and Hong-fu ZHANG declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1400266 Project supported by the Chinese National Science and Technology Pillar Program (No. 2012BAD39B0) and the Special Fund for Innovation Team of the Chinese Academy of Agricultural Sciences (No. ASTTP-IAS07) 2014-10-03 2015-05-13 2015-01-31 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 16