Proteomic analysis reveals that lysine acetylation mediates the effect of antemortem stress on postmortem meat quality development

Food Chemistry 293 (2019) 396–407

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Proteomic analysis reveals that lysine acetylation mediates the effect of antemortem stress on postmortem meat quality development ⁎

Bing Zhoua,b, Zhenglei Shenc, Yisong Liub, Chengtao Wanga, , Qingwu W. Shena,b,

T

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a

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100048, China College of Food Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, China c Nanya Middle School, Changsha, Hunan 410129, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Proteomics Protein lysine acetylation Antemortem stress Meat quality Glycolysis

To explore the involvement of protein lysine acetylation in the conversion of muscle to meat, a quantitative analysis of the acetylome in postmortem porcine muscle with or without antemortem stress was conducted. In total, 771 acetylpeptides containing 681 lysine acetylation sites mapping to 176 acetylproteins were identified. Acetylproteins were enriched in muscle contraction, carbohydrate metabolism, cell apoptosis and calcium signaling. Bioinformatic analysis suggests that preslaughter handling may be associated with glycolysis in postmortem muscle and the overall meat quality, via acetylation of multiple enzymes of glycogenolysis/glycolysis, regulate rigor mortis via acetylation of contractile, ATP production and calcium signaling-related proteins, and regulate stress response, cell apoptosis and meat tenderization via regulating the functions of heat shock proteins and permeability transition pore complex. This study provides the first overview of the acetylome in postmortem muscle as affected by preslaughter handling and broadens knowledge of the biochemistry regulating meat quality development.

1. Introduction The postmortem (PM) conversion of muscle to meat involves a series of physico-biochemical changes, including pH decline, programmed cell death or apoptosis, rigor mortis, proteolysis and so on. Among these changes, the decline of muscle pH resulting from glycolysis and lactate accumulation is probably the most important factor determining meat quality, as it influences or relates to most of the meat quality indicators, such as meat water-holding capacity, color and flavor (Scheffler & Gerrard, 2007; Wulf, Emnett, Leheska, & Moeller, 2002). Abnormal glycolysis and pH decline in PM muscle lead to the development of PSE (pale, soft, and exudative), DFD (dark, firm, and dry) or acid meat, which is not preferred by consumers, due to its inferior quality (Scheffler & Gerrard, 2007; Shen & Du, 2015). According to surveys, the PSE and DFD problems cause the meat industry an annual loss of millions of dollars in the United states (Vansickle, 2006). In addition to glycolysis, the PM progress of apoptosis, rigor mortis and proteolysis all influence meat tenderization and its commercial value. Numerous studies aiming to improve meat quality have identified that pre-slaughter stress and genetics are the two main factors

contributing to the increased occurrence of PSE and DFD meat (Leheska, Wulf, & Maddock, 2002; Rosenvold & Andersen, 2003; Shen & Du, 2015). Pre-slaughter factors include transport, lairage, mixing of unfamiliar animals, stunning methods, and slaughter conditions. Generally, short term acute preslaughter stress increases glycolytic rates early postmortem and the PSE risk in pigs and poultry. Resting for animals to recover from stress before slaughter is beneficial and improves the final quality of raw meat. For example, one hour transport right before slaughter increases the drip loss and PSE likelihood of pork loins, while two hour resting after transport significantly reduces the adverse effect of transport (Shen, Means, Thompson, et al., 2006). Some researchers have also reported that lairage time is related to the progress of rigor mortis and muscle tension at 3 h PM, which may influence meat tenderness (Dokmanovic et al., 2015). Despite these observations, the molecular mechanism underlying these phenomena, especially the action of preslaughter factors on protein functions in PM muscle at proteome levels, is not well understood. The protein acetylation at ε-NH2 of lysine residue (termed lysine acetylation) is a common post-translational modification that may rival phosphorylation (Kouzarides, 2000). Although it has been discovered

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Corresponding authors at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, No. 11 Fucheng Road, Haidian District, Beijing 100048, China (C. Wang). College of Food Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, China (Q.W. Shen). E-mail addresses: [email protected] (C. Wang), [email protected] (Q.W. Shen). https://doi.org/10.1016/j.foodchem.2019.04.122 Received 16 February 2019; Received in revised form 17 April 2019; Accepted 30 April 2019 Available online 02 May 2019 0308-8146/ © 2019 Published by Elsevier Ltd.

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pork loin chops were dissected from the same location on the right side of the carcasses and used for meat quality measurement.

for a long time (Phillips, 1963), the lack of high-throughput technologies for acetylprotein identification greatly limited studies in this area, although histone acetylation and its regulation of gene expression have been intensively studied and well recognized in the past decades (Ali, Conrad, Verdin, & Ott, 2018). The invention of the anti-acetyllysine antibody in combination with the development of mass spectrometrybased proteomics strategy has greatly advanced our understanding of protein acetylation in biological processes. Currently, > 100 systemwide proteomic analyses have identified thousands of lysine acetylation sites distributed in > 1500 proteins located in virtually every cellular compartment (Ali et al., 2018; Choudhary et al., 2009; Schneider, Kramer, Schmid, & Saur, 2011). Protein acetylation has been recognized to regulate enzyme activities, protein stability, cell signaling, and even diseases (Ali et al., 2018; Montgomery, Sorum, & Meier, 2015; Spange, Wagner, Heinzel, & Kramer, 2009). Most enzymes involved in glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid oxidation, and oxidative phosphorylation are acetylated and protein lysine acetylation/deacetylation regulates cellular metabolism (Chen et al., 2018; Choudhary et al., 2009; Wang et al., 2010; Zhao et al., 2010). Thus, it is hypothesized that protein lysine acetylation regulates glycolysis, rigor mortis, and/or proteolysis in PM muscle and the final meat quality. In addition, we further hypothesized that protein lysine acetylation/deacetylation mediates the influence of preslaughter handling on meat quality development. Indeed, our previous study has revealed that antemortem stress regulates protein acetylation and glycolysis in postmortem muscle (Li, Li, Wang, Shen, & Zhang, 2016). By using histone acetyltransferase (HAT) and deacetylase (HDAC) inhibitors, we have further found that protein acetylation is involved in AMP-activated protein kinase (AMPK) regulation of glycolysis in PM muscle (Li et al., 2017). However, no further information is available regarding the specific acetylation sites and acetylproteins mediating the effect of antemortem stress on meat quality development. The purpose of this study was to comparatively profile protein lysine acetylation in PM porcine muscle with different preslaughter handlings to screen the candidate acetylproteins/lysine acetylation sites that mediate the influence of antemortem stress on meat quality and/or play an important regulatory role in PM meat quality development, which could be used to control or improve meat quality in the future. The data obtained showed that preslaughter handling had a complicated influence on protein lysine acetylation in PM porcine muscle. It suggested that antemortem stress might regulate the PM conversion of muscle to meat and the final quality of raw meat via dynamic acetylation/deacetylation of multiple proteins involved in cell apoptosis, glycogenolysis/glycolysis, muscle contraction and calcium signaling. This is the first acetylproteomic survey of PM muscle aimed to explore and provide some new insights into the mechanisms by which antemortem stress influences meat quality

2.2. Meat quality measurement Muscle pH was measured on the carcass by directly inserting the electrode of a portable pH meter (HI99161, Hanna Instruments Inc., Italy) with temperature compensation into muscle. Lactate in muscle was determined using a commercial assay kit according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China). Meat surface color (CIE L*a*b*), drip loss, cooking yield and shear force were measured as in the literature (Shen, Means, Underwood, et al., 2006). 2.3. Protein extraction, trypsin digestion and acetylpeptide enrichment For proteomic analysis, the 6 pigs within each treatment were randomly paired and equal mass of LD muscles from each pig was powdered and mixed in liquid nitrogen to obtain three biological replicates. Muscle (0.2 g) was sonicated on ice in 1 mL ice-cold lysis buffer containing 8 M urea and complete protease inhibitor (Roche, Cat#:04693116001, one tablet per 50 mL) for 60 s with 0.2 s on and 2 s off by using a SCIENTZ-II D sonicator (Ningbo Xinzhi Biotechnology Co., Ltd, Ningbo, China). To inhibit endogenous protein deacetylase, 10 μM trichostatin A, 10 mM nicotinamide and 50 mM sodium butyrate were added to lysis buffer. After extraction on ice for 30 min, samples were centrifuged at 15,000 g, 4 °C for 20 min. The supernatant was carefully collected to avoid the fat layer and protein concentrations were determined using the Bradford method (Thermo Scientific, Cat#: 23238). Trypsin digestion of proteins and immunoaffinity enrichment of acetylated peptides were conducted as previously described (Chen et al., 2018; Choudhary et al., 2009) with some modification. Briefly, 10 mg of extracted proteins were added with 1 M dithiothreitol (DTT) and reduced in 10 mM DTT for 60 min at 37 °C. The reduced proteins were then added with 1 M freshly made iodoacetamide (IAA) to a final concentration of 40 mM and alkylated for 40 min at room temperature in the dark. The protein sample was diluted using 100 mM NH4CO3 to a urea concentration < 2 M and digested overnight at 37 °C using sequencing grade trypsin (Promega Corporation, Madison, WI; Cat#: V5111) with a trypsin/protein ratio of 1/50. Samples were heated at 99 °C for 5 min to deactivate trypsin, desalted on C18 column and lyophilized. Lysine acetylated peptides were enriched using the agarose-conjugated anti-acetyllysine antibody (PTMScan kit; Cell Signaling Technology, London, UK; Cat#: 13416). Briefly, the antibody beads were prepared by washing twice with phosphate buffered saline (PBS) and then twice with immunoaffinity purification (IAP) buffer (50 mM MOPS, pH 7.2, 10 mM sodium phosphate, 50 mM NaCl). Tryptic peptides dissolved in IAP buffer were added with washed beads (100 μL beads per 10 mg protein) and incubated at 4 °C for 2 h with gentle rotation. The beads were collected by centrifugation (2000 g, 30 s, 4 °C), washed twice with IAP buffer and then three times with ice-cold ddH2O. The beads were added with 55 μL of 0.15% trifluoroacetic acid (TFA) and incubated at room temperature for 10 min to elute bound peptides. After centrifugation at 2000 g, 4 °C for 30 s, the supernatant containing acetylpeptides was collected and the elution procedure was repeated with 50 μL of 0.15% TFA. The eluted peptides were combined, desalted and vacuum dried as described above.

2. Materials and methods 2.1. Animals and muscle collection Pigs were handled and muscles were collected as previously described (Shen, Means, Thompson, et al., 2006). Briefly, eighteen crossbreed (Duroc × Landrace × Yorkshire) pigs with an average weight of approximately 110 kg were randomly assigned to three treatments: (1) Control pigs were delivered to abattoir 12 h prior to slaughter; (2) Transport (stressed), pigs were transported for one hour to be stressed before slaughter; (3) Rest (recovered, lairage), pigs were transported for one hour and then rested for two hours before slaughter. Pigs were slaughtered according to the commercial procedure at a commercial slaughter house (Hunan Weihong Co., Ltd, Xiangtan, China). The longissimus dorsi (LD) muscle between the 10th and 11th thoracic vertebrae on the left side of the carcasses was sampled at 0 (immediately following exsanguination), 0.5, 1, 4, and 24 h PM, snap-frozen in liquid nitrogen and stored at −80 °C until analysis. At 24 h PM, two boneless

2.4. LC-MS/MS analysis The enriched peptides were dissolved in 2% methanol with 0.1% formic acid and separated on a reversed-phase analytical column (EASY-Spray™ column, C18, 75 μm × 12 cm, 3 μm) equipped with a reversed-phase precolumn (Acclaim PepMap100 column, C18, 397

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2.39 ± 0.06b 3.43 ± 0.11a 3.20 ± 0.21a

2.5. Data search and peptide quantification The LC–MS/MS data were processed with MaxQuant software (version 1.5.2.8) and subjected to database searching against the UniProt Sus Scrofa (pig) protein database concatenated with reverse decoy database with the following criteria: enzyme, trypsin; maximum missed cleavages, 2; static modifications, carbamidomethylcysteine (57.021 Da); dynamic modifications, oxidation (M) and acetylation (K, protein N-terminus, KCSYHT). Mass error was set as 5 ppm for precursor and 0.02 Da for fragments. False discovery rate (FDR) thresholds for protein, peptide and modification site were all set at 0.01. Only acetylation identifications with a localization probability ≥0.75 were considered acetylation sites. Peptides with different amino acid sequences or acetylation sites were considered unique. The MS spectra were used to determine the identity and abundance of each unique peptide. To calculate the relative abundance of a peptide, the MS intensities of all identified acetylpeptides of each sample was first normalized against the median which was set as 1 after removing peptides with intensity of 0. The ratio of MS intensity was then calculated to compare the relative abundance of a peptide between samples with a ratio > 1.5 or < 0.67 defined as significant. Nine (3 × 3) comparisons were performed for the six biological replicates between two treatments at the same sampling time points. Results were reported as up-regulated, down-regulated or no change if ≥5 of the 9 comparisons showed the same change pattern. The relative acetylation levels of a specific site (lysine residue) were evaluated by the relative abundances of acetylpeptides identified for the site. The evaluation of the acetylation of a protein was based on the relative acetylation levels of all lysine sites identified in the protein. The up-regulated or downregulated acetylation sites within the protein were required to be more than both the opposite regulated sites and the unchanged sites, if the acetylation of the protein was reported to be up-regulated or downregulated. Nine comparisons were performed between two treatments for acetylproteins as done for unique peptides.

6.33 ± 0.06 5.99 ± 0.10b 6.24 ± 0.08ab 6.43 ± 0.06 6.13 ± 0.08b 6.32 ± 0.05ab 6.50 ± 0.09 6.35 ± 0.06a 6.37 ± 0.05a Control Transport Rest

2.6. Bioinformatics analysis Functional classification and enrichment of identified acetylproteins based on the Gene Ontology (GO) terms, including cellular component, molecular function and biological process, were performed using the DAVID software (http://david.abcc.ncifcrf.gov/). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www. kegg.jp/) was used to identify enriched pathways. Porcine genes were chosen as the background population for all analyses. The corrected pvalue < 0.05 was considered as significant for all of the bioinformatics

a,b,c

54.56 ± 1.32ab 56.30 ± 0.73a 52.45 ± 0.88b 5.68 ± 0.02a 5.68 ± 0.01a 5.74 ± 0.04a 6.04 ± 0.09 5.66 ± 0.03b 5.86 ± 0.08a

24 h

a

4h

a

1h

a

0.5 h

a

0h

Within the same column, means lacking a common superscript letter differ significantly (p < 0.05).

16.14 ± 0.91a 16.76 ± 0.68a 17.57 ± 0.50a

9.55 ± 0.65a 10.47 ± 0.37a 10.09 ± 0.46a

3.4 ± 0.5b 7.4 ± 1.6a 4.1 ± 1.1ab

Drip loss (%) Shear force (kg) Yellowness (b*) Redness (a*) Lightness (L*) Muscle pH values Treatments

Table 1 Some indicators of meat quality.

100 μm × 2 cm, 5 μm) using an EASY-nLC 1000 UPLC system (all Thermo Scientific). Solvent A was 0.1% formic acid. Solvent B was acetonitrile (ACN) with 0.1% formic acid. The separation was performed in gradient mode: an increase of solvent B from 4% to 15% in 5 min, then to 25% in 35 min, 35% in 25 min and finally to 95% in 5 min. Solvent B was held at 95% for 12 min and quickly decreased to 4% in 3 min. The column was washed with 4% solvent B for 5 min before the next separation. The peptides were subjected to a nanospray ionization source and tandem mass spectra were collected in a data-dependent manner with an Orbitrap Fusion mass spectrometer (Thermo Scientific). The applied electrospray voltage was 2.0 kV. Capillary temperature was set at 250 °C. A survey MS scan (m/z 350–1800) was acquired in the Orbitrap analyzer with a resolution of 70,000 and an automatic gain control target of 1 × 106 (maximum ion time: 60 ms). Peptides were selected for MS/MS using an NCE setting of 29. The top most intense 20 ions were defaulted for higher energy collisional dissociation MS/MS spectra in the ion trap with a resolution of 17,500 and an automatic gain control target of 5 × 106 (maximum ion time: 70 ms). Ion intensity threshold was set at 5000.

83.6 ± 1.2a 75.8 ± 2.2b 81.1 ± 1.9ab

Cooking yield (%)

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and acetylproteins were commonly identified in the three treatments, for example, 426, 274, 283, 310 and 99 acetylation sites along with 114, 85, 84, 98 and 41 acetylproteins were commonly identified in the muscles from control, stressed and recovered pigs at 0, 0.5, 1, 4 and 24 h PM, respectively (Fig. S1); (2) the numbers of both identified acetylation sites and acetylproteins totally decreased from 0 to 24 h PM, but a temporary increase from 1 to 4 h PM in the muscles from control and stressed pigs and an increase from 0.5 to 1 h PM in the muscles from rested pigs were determined (Fig. 1); (3) at the very early stage within 0.5 h PM, the acetylproteins specifically commonly expressed between the control and stressed pigs (5 acetylproteins at 0 h and 4 acetylproteins at 0.5 h PM) were fewer than those specifically commonly expressed between the control and rested (10 acetylproteins at 0 h and 15 acetylproteins at 0.5 h PM) or the stressed and rested pigs (9 acetylproteins at 0 h and 11 acetylproteins at 0.5 h PM). The differences in the time course change patterns of protein acetylation in PM muscle and the numbers of commonly expressed acetylproteins between different treatments reflected the effect of different preslaughter handlings on protein acetylation in PM porcine muscle. The distribution of the numbers of acetylation sites per protein are shown in Fig. 1D. More than half of the identified acetylproteins in porcine muscle were mono-acetylated. About 12% of the total identified acetylproteins were di-acetylproteins. About 6% of the identified acetylproteins were detected to have 3 lysine acetylation sites and another 6% had 4 acetylation sites. In addition, about 20% of the total identified acetylproteins had 5 or more lysine residues acetylated. The highest number of lysine acetylation sites was detected in myosin heavy chains (Table S4), although some sequences are commonly shared by the different isoforms of myosin heavy chains and it is difficult to determine the exact number of acetylation sites in each isoform. In the present study, 80, 44 and 38 acetylation sites were assigned to the intermediate myosin heavy chain 2× (MHC-2×, gene name MYH1), the slow type MHC-β (MYH7) and the fast type MHC-2b (MYH4), respectively. Multiple lysine acetylation sites were also detected in some other myofibrillar proteins, like myomesin, tropomyosin, actin, actinin and so on. Following sarcomere proteins, enzymes involved in glycolysis and glycogenolysis were acetylated at multiple locations, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, glycogen phosphorylase (GP), pyruvate kinase (PK), lactate dehydrogenase (LDH) and others.

analysis. Differentially acetylated proteins were subjected to protein–protein interactions analysis by searching against the STRING database (Search Tool for the Retrieval of Interacting Genes/Protein, http://string-db. org/) with a high confidence score (score ≥0.7), the interaction networks showed only the proteins with connections. The unconnected proteins were not presented. 3. Results 3.1. The effect of antemortem stress and resting on meat quality To investigate the effect of pre-slaughter stress on meat quality, we used one hour transport to stress pigs and two hour resting after transport for pigs to recover from stress before exsanguination. The effect of these pre-slaughter handlings on meat quality and some carcass characteristics are shown in Table 1 and S1. As previously reported, pre-slaughter transport or stress had an obviously adverse effect on pork quality. Transport of pigs before slaughter significantly increased the shear force and drip loss of meat, and decreased cooking yield when compared to the control, which is probably associated with the increased likelihood of PSE meat (Fortin, 2002; Shen, Means, Thompson, et al., 2006). Indeed, lower muscle pH values and higher lactate concentrations in muscle from 0.5 to 4 h PM, and higher carcass temperature within 1 h PM were determined in the carcasses of transported pigs. As expected, two hours of rest for pigs to recover from stress had a beneficial effect on meat quality. It returned the waterholding capacity (WHC) of pork loin to a similar level to the control, though meat from these pigs still had tougher texture when compared to the control. In addition, resting slowed down the glycolytic rate in muscle early postmortem, as the muscle pH values of the rested pigs were not different from those of the control pigs. It is worth mentioning that meat from the rested pigs had darker surface color (p < 0.05), when compared to the transport group. Its L* value was also numerically lower than that of the control, though not statistically different, showing the effect of extended lairage time on meat lightness, as previously reported (Dokmanovic et al., 2015). In summary, these data showed that pre-slaughter transport and resting significantly altered glycolysis in PM muscle and the final meat quality, and thus the collected muscle samples were suitable for subsequent proteomic analysis with regard to the purpose of the present study.

3.3. Functional classification and enrichment of acetylproteins in PM porcine muscle

3.2. Profiling of lysine acetylation sites and acetylproteins in PM porcine muscle

To have an overview of the events that protein acetylation was involved in, all acetylproteins identified in PM porcine muscle were subjected to GO analysis using the DAVID software. As shown in Fig. 2, membranal, mitochondrial and cytoplasmic acetylproteins were highly enriched in PM porcine muscle, followed by acetylproteins located in the cytoskeleton, extracellular region, nucleus, peroxisome, endoplasmic reticulum (ER) and Golgi apparatus. In the molecular function ontology, acetylproteins with signal transducer activity, metal ion binding, transmembrane transporter activity and rRNA binding activity were enriched in the present study. GO enrichment on the classification of biological processes found that acetylproteins in porcine muscle were highly enriched in carbohydrate metabolism, glucose metabolism, and signal transduction, followed by acetylproteins in immune system process, transmembrane transport, cell death, transport, reproduction, plasma membrane organization, and cell adhesion. In addition, KEGG pathway identification showed that acetylproteins in porcine muscle were enriched in carbon fixation, biosynthesis of amino acids, biosynthesis of antibiotics, 2-oxocarboxylic acid metabolism, Parkinson's disease, Huntington's disease, glycolysis/gluconeogenesis, pentose phosphate pathway, calcium signaling pathway, and fructose and mannose metabolism. These data showed that protein lysine acetylation was involved in glucose metabolism, calcium signaling and cell death,

In total, 771 acetylpeptides containing 681 acetylation sites distributing to 176 proteins were identified in porcine LD muscle in the present study. The numbers of identified acetylpeptides, lysine acetylation sites and acetylproteins in each treatment at different PM times are illustrated in Fig. 1 and S1. Detailed information about the identified acetylpeptides, lysine acetylation sites and acetylproteins are listed in Tables S2–S4. In the muscle from the control pigs, 590, 408, 382, 448 and 180 acetylpeptides containing 522, 373, 357, 397 and 153 acetylation sites mapping to 139, 112, 100, 117 and 48 proteins, respectively, were identified at 0, 0.5, 1, 4 and 24 h PM. Most of the peptides were mono-acetylpeptides and only 5 were di-acetylpeptides. No acetylpeptides were identified to have three or more lysines acetylated in all three groups. In the muscle from the stressed pigs, 591, 402, 373, 440 and 156 acetylpeptides with 520, 369, 360, 410 and 141 acetylated lysines and mapping to 133, 103, 104, 124 and 50 proteins, respectively, were identified at 0, 0.5, 1, 4 and 24 h PM. In the muscle from the rested pigs, 613, 413, 441, 414 and 138 acetylpeptides containing 533, 380, 406, 389 and 124 acetylation sites mapping to 142, 118, 121, 113 and 48 proteins, respectively, were identified at 0, 0.5, 1, 4 and 24 h PM. Based on the acetylation sites and acetylproteins identified in PM porcine muscle, it could be generalized: (1) most acetylation sites 399

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Fig. 1. Lysine acetylation sites and acetylproteins identified in PM muscle of pigs with different preslaughter handling. A–C, the numbers of acetylpeptides, lysine acetylation sites and acetylproteins identified in PM muscle of differently handled pigs. D, Pie chart illustration of the numbers of lysine acetylation sites per protein.

collected at 0 h PM had the highest total acetylation levels and grouped into the second cluster, of which T1 (Transport, 0 h PM) was more closely related to R1 (Rest, 0 h PM) than to C1 (Control, 0 h PM). T2 (Transport, 0.5 h PM), R2 (Rest, 0.5 h PM), R4 (Rest, 4 h PM), C3 (Control, 1 h PM) and T3 (Transport, 1 h PM) were grouped into the third cluster with T2 closely related to R2 and C3 closely related to T3. C2 (Control, 0.5 h PM), C4 (Control, 4 h PM), R3 (Rest, 1 h PM) and T4 (Transport, 4 h PM) were grouped into the fourth cluster with R3 closely related to T4. Hierarchical clustering also showed that the total protein lysine acetylation was more similar between the Transport and Rest groups at 0 and 0.5 h PM when compared to the control. At 1 h PM, multiple sites in the rested muscle (R3) were up-regulated in total acetylation compared to the other two treatments (C3 and T3) and R3 was grouped in the same cluster with C4 and T4. In summary, these data show that preslaughter handling altered protein lysine acetylation in PM muscle, which may be a reason for the different meat quality via its regulation of glycolysis, muscle contraction and rigor mortis, and other events important to meat quality development, such as cell apoptosis and calcium signaling.

which were critical to the PM development of meat quality. 3.4. Quantification of protein lysine acetylation in PM porcine muscles To explore the influence of pre-slaughter handling on protein lysine acetylation in PM muscle, the relative acetylation levels of specific acetylation sites were evaluated based on the MS intensity with a ratio > 1.5 or < 0.67 defined as significant. Compared to the control pigs, 107, 96, 59 39 and 65 acetylation sites were down-regulated (MS intensity ratio < 0.67) and concomitantly 96, 82, 73, 118, and 11 acetylation sites were up-regulated (MS intensity ratio > 1.5) in the stressed pigs at 0, 0.5, 1, 4, and 24 h PM, respectively (Fig. 3A). In the muscle from pigs rested for two hours after transport, 131, 104, 55, 80, and 42 sites were down-regulated and 112, 91, 157, 63, and 20 sites were up-regulated at 0, 0.5, 1, 4, and 24 h PM, respectively, when compared to the control. Two hour resting also let to 102, 65, 43, 137, and 10 acetylation sites down-regulated and 78, 44, 145, 50, and 41 sites up-regulated in porcine muscle at 0, 0.5, 1, 4, and 24 h PM, respectively, when the two transported groups were compared. Correspondingly, preslaughter transport and rest also led to multiple proteins differentially acetylated between treatments (Fig. 3B). Generally, more acetylation sites and acetylproteins were up-regulated than downregulated in the stressed muscle at 1 and 4 h PM when compared to the control, and more acetylation sites and acetylproteins were up-regulated than down-regulated in the rested muscle at 1 h PM, when compared to both the control and stressed muscles. Hierarchical clustering grouped porcine muscle samples into 4 clusters according to the acetylation levels of the 66 lysine acetylation sites that were identified in all three biological replicates. Muscles collected at 24 h PM from the control (C5), transported (T5) and rested pigs (R5) had the lowest acetylation levels and grouped into the first cluster (Fig. 4). All muscles

3.5. Protein-protein interaction networks of differentially acetylated proteins To better understand the involvement of protein lysine acetylation in the regulation of meat quality in response to preslaughter handling, differentially acetylated proteins were subjected to protein–protein interactions analysis by searching against the STRING database with a high confidence score (score ≥ 0.7). At 0 h PM, 3 networks were identified for the differentially acetylated proteins in the control and stressed muscle (Fig. 5), which were networks of TCA cycle related proteins (CS, DLD, IDH3A, DLST-tv1, SUCLA1, ACAT1 and ALDH4A1), 400

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Fig. 2. Functional classification and enrichment of acetylproteins identified in PM porcine muscle. A–C, GO function classification analysis of total identified acetylproteins in PM porcine muscle according to their cellular component (A), molecular function (B) and biological process. D, KEGG pathway enrichment analysis of total identified acetylproteins in PM porcine muscle.

Fig. 3. The numbers of up-regulated and down-regulated lysine acetylation sites and acetylproteins in PM porcine muscle. The relative acetylation levels of an acetylation sites were evaluated by the MS intensity of acetylpeptides identified for the site. A ratio > 1.5 or < 0.67 was defined as up-regulated or down-regulated. The relative acetylation level of a protein was determined by the relative acetylation levels of all acetylation sites identified for the proteins as described in Materials and Methods. C1 means control group at 0 h PM. C2 means control group at 0.5 h PM. C3 means control group at 1 h PM. C4 means control group at 4 h PM. C5 means control group at 24 h PM. The same labeling was used for the Transport (T) and Rest (R) treatments. Sig (T1/C1) means the comparison of T1 to C1 (and the same lettering was used for the other comparisons). 401

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Fig. 4. Hierarchical clustering of muscle samples according to the acetylation levels of 66 lysine acetylation sites identified in all muscle samples.

the networks of ATP synthase and muscle contractile proteins, a large network of glycogenolysis/glycolysis (PYGB, PGM1, PFKM, ENO1, GPI, PKM and DLD) was identified for the differentially acetylated proteins in the stressed and control muscles at 0.5 h PM. The network of glycogenolysis/glycolysis could be a reason for the different glycolytic

muscle contraction related proteins (TPM2, MYBPC1, MYBPC2, MLC3F, NEB, and DES), and ATP synthesis and apoptosis related proteins (ATP5A1, ATP5B and SLC25A5). This could be explained by the increased aerobic metabolism and ATP production, as well as muscle contraction, induced by preslaughter transport and stress. In addition to 402

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Fig. 5. Protein-protein interaction networks of differentially expressed acetylproteins in PM porcine muscle. Nodes of triangles in red represent up-regulated and nodes of V shapes in green represent down-regulated acetylproteins. Edge stroke colors show reported interactions based on experimental evidence (red) or database (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

1 h PM, most acetylproteins identified in the network searching were up-regulated in the rested muscle when compared to the control, which should be related to the greater muscle tension of the Rest group, as lairage time affects the onset of rigor mortis and muscle tension (Dokmanovic et al., 2015). When the two transported groups were compared, a network of contractile proteins (MYBPC1, DES, ACTA2, TMP2 and ACTA1), a small network of three glycolytic enzymes (ENO1, GPI and ALDOC), and three proteins in the electron transport chain were identified at 0 h PM (Fig. 5). At 0.5 h PM, no network of glycolysis and only a small network of electron transport and oxidative phosphorylation were identified for the Transport and Rest groups, which was consistent with the muscle pH values that were not different between these two groups early PM. At 1 h PM, a network of TCA cycle was identified and acetylproteins in the network were all up-regulated in the rested muscle. On the contrary, most acetylproteins in the identified networks were down-regulated in the rested muscle when compared to the control at 4 h PM, including networks of glycolysis, aerobic metabolism, stress response and apoptosis, and muscle contraction (Fig. 5).

rates between these two treatments at early PM stage. At 1 h PM, several proteins related to muscle contraction (NEB, MYBPC1, MLC3F, TPM2, MYH7B and TPM3), glycogenolysis/glycolysis (ALDOC, PGM1 and PYGM), TCA cycle (DLST-tv1, DLD, ALDH4A1, and GOT1), and apoptosis (SLC25A6, VDAC1 and VDAC2) were differentially acetylated in the control and stressed muscles. The network of apoptosis suggests that antemortem stress influenced the progress of cell death after exsanguination. At 4 h PM, most of the differentially acetylated proteins were up-regulated in the stressed muscle when compared to the control. These differentially expressed acetylproteins were involved in glycogenolysis/glycolysis, TCA cycle, muscle contraction and cell apoptosis. When compared between the Control and Rest groups, a network of glycolysis/TCA cycle (ALDOC, ENO1, GPI, CS, ADSL, IDH2, ACO2, SUCLA2, ACAT1, and DLST-tv1) was identified for these two treatments at 0 h PM (Fig. 5). In addition, two proteins (VDAC2 and SLC25A6), which are components of permeability transition pore complex (PTPC, implicated in cell death), and 4 sarcomere proteins (MYH2, ACTA2, MYH3 and MYBPC1) were identified in the protein–protein interaction network searching. These differentially acetylated proteins reflected that pigs in the Rest group were still stressed when compared to pigs in the Control group. This is logical because pigs in the Rest group were transported for one hour and waited in lairage for two hours before slaughter. At 0.5 h PM, no network of glycolysis was identified for the control and rested pigs. This explained why the glycolytic rates and muscle pH values were not different between these two treatments. At

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mechanism by which preslaughter stress regulates glycolysis in PM muscle remains largely unclear. Recently, our studies revealed that protein lysine acetylation was involved in the regulation of postmortem glycolysis by preslaughter stress and AMPK as manipulation of protein lysine acetylation by HAT and HDAC inhibitors altered glycolysis in PM muscle (Li et al., 2017, 2016). However, no information is available about the exact enzymes or proteins that are regulated by lysine acetylation and subsequently control glycolysis in PM muscle. For this reason, here we comparatively profiled protein lysine acetylation in PM muscles from pigs differently handled prior to slaughter. In agreement with the literature (Du, Shen, & Zhu, 2005; Shen & Du, 2005; Shen, Means, Thompson, et al., 2006), lower pH values and higher lactate concentrations were determined 0.5–4 h PM in the muscle from pigs transported for one hour when compared to the control. At the same time, a network of glycolysis/glycogenolysis was identified using the differentially acetylated proteins between these two treatments at 0.5 h PM. In the network, glycogen phosphorylase (PYGB), phosphoglucosemutase (PGM1), phosphoglucose isomerase (GPI), enolase (ENO1), pyruvate kinase (PKM), and dihydrolipoyl dehydrogenase (DLD) were down-regulated but phosphofructokinase (PFKM) was up-regulated in stressed muscle. These differently expressed acetylproteins are probably responsible for the increased glycolytic rate in the stressed muscle. When protein acetylation in rested muscle was quantitatively compared to that in the control muscle, a small network of glycolysis, composed of GPI, ENO1 and aldolase (ALDOC), was identified at 0 h PM for these two treatments. This should be an illustration of the in vivo difference in glycolysis which was induced by the extended lairage time (2 h resting). In agreement with muscle pH and lactate concentrations, glycolytic enzymes were not differentially acetylated in rested and control muscle at 0.5 h PM, but two enzymes, PYGB and PGM1, which are involved in glycogen utilization, were down-regulated. These data further suggest that protein acetylation/deacetylation was involved in the regulation of postmortem glycolysis. The decreased acetylation of PYGB and PGM1 probably upregulated glycogenolysis in the rested muscle as a result of glucose depletion induced by extended lairage time. Two hour resting after transport also led to glycolytic enzymes, GPI, ENO1 and ALDOC, differentially acetylated between the Transport and Rest groups at 0 h PM. No network of glycolysis or glycogenolysis was identified for these two treatments at 0.5 h PM. This is consistent with the measured lactate content in muscle, which was not different between the two treatments, and muscle pH, which was not different before 4 h PM. Hexokinase, phosphofructokinase and pyruvate kinase are traditionally believed to be the rate-limiting enzymes in glycolysis. Hexokinase was not identified to be acetylated in porcine muscle in the present study. This is in agreement with the finding that no literature has ever reported a rate-limiting role of hexokinase in PM glycolysis. In addition to phosphofructokinase and pyruvate kinase, two non-ratelimiting enzymes, phosphoglucose isomerase (GPI) and enolase 1 (ENO1) were found to be differentially acetylated between the Transport and Control groups at 0.5 h PM. These data suggest that the classically non-rate-limiting enzymes may play an important regulatory role in postmortem glycolysis. Indeed, a combined proteomic and enzyme kinetic study revealed that the rate controlling enzymes in glycolysis are tissue specific. Some classically non-rate-limiting enzymes determined in liver, such as aldolase, phosphoglycerate kinase, or enolase are probably rate-limiting in skeletal muscle (Wisniewski, Gizak, & Rakus, 2015). In addition, many conflicting reports (Scheffler & Gerrard, 2007), regarding the relationship between enzyme activity and muscle pH as well as PSE risk, suggest that a distinct regulatory mechanism may exist and classically non-rate-limiting glycolytic enzymes may become rate-limiting in PM muscle. In the present study, the glycolytic rates were not different between the Rest and Control group, but the two enzymes in glycogenolysis, PYGB and PGM1, were determined to be differentially acetylated at 0.5 h PM. This could be a response to the accelerated depletion of glucose in the rested muscle and a case of

4.1. Preslaughter handling, pork quality and protein lysine acetylation in PM muscle Many studies have reported that stress induced by preslaughter transport results in adverse meat quality (Rosenvold & Andersen, 2003; Shen & Du, 2015). A survey of over 1900 pigs by Fortin (Fortin, 2002) reported that pigs transported for 50 min before slaughter had the highest incidence of PSE meat compared to other treatments and a 3-h resting period significantly improved meat quality by reducing the occurrence of PSE loins. In addition, our previous study also showed that one hour transport before slaughter increased PSE risk by decreasing muscle pH early postmortem and meat WHC and increasing meat lightness (Shen, Means, Thompson, et al., 2006). It has also been reported that two hour resting in lairage is sufficient to alleviate the adverse effect of transport and significantly improves meat quality (Fortin, 2002; Owen, Montgomery, Ramsey, & Miller, 2000; Shen, Means, Thompson, et al., 2006). In agreement with the literature, the data obtained in the present study showed that one hour transport before slaughter significantly increased meat drip loss and decreased cooking yield, which increased from 3.4% to 7.4% and decreased from 83.6% to 75.8% in the Control and Transport groups (Table 1; p < 0.05), respectively, showing the detrimental effect of transport on meat WHC. Two hour resting reversed the bad effect of transport on meat WHC. However, pork loin from both the Transport and Rest groups had higher shear force, showing tougher texture and lower tenderness of meat. As pigs in these two groups were all transported for one hour before slaughter, this result showed that preslaughter transport had an adverse effect on meat tenderness. In addition, meat from the Rest group was relatively much darker than from the other two groups, showing extended lairage time had an influence on meat surface lightness. Totally 771 acetylpeptides containing 681 lysine acetylation sites distributing to 176 acetylproteins were identified in the present study. Functional enrichment and protein–protein interaction network analysis revealed that acetylated proteins in PM muscle were enriched in muscle contraction, glycogenolysis/glycolysis, cell death and calcium signaling, which are related to cell death, pH decline and rigor mortis in PM muscle that are critical to meat quality development. Quantification of the numbers of both identified lysine acetylation sites and acetylproteins showed that protein lysine acetylation in all three treatments was totally down-regulated with increasing PM time, but a temporary increase in both acetylation site and acetylprotein numbers was determined in the control and stressed muscle at 4 h PM and in the rested muscle at 1 h PM. The up-regulation of protein acetylation within 1–4 h PM compared to an earlier time point was probably related to the onset of rigor mortis happening during this period in porcine muscle. In addition, most identified acetylproteins related to muscle contraction and glycogenolysis/glycolysis were acetylated at multiple lysine residues. Quantification of acetylation at specific lysine residues and protein levels showed that preslaughter handling had a complicated influence on protein lysine acetylation in PM muscle, which was acetylation site and PM time dependent. In accordance with our hypothesis, these results demonstrated that protein lysine acetylation might play a role in the conversion of muscle to meat, which was regulated by and may mediate the effect of preslaughter handling on meat quality. 4.2. Antemortem stress, protein acetylation and glycolysis in PM muscle Low pH resulted from fast and excessive glycolysis in PM muscle, especially at early stages when the muscle temperature is high, denaturing muscle proteins, which is the cause of PSE meat. Many studies have reported that antemortem stress increases glycolytic rate in PM muscle, which is the reason for increased risk of PSE meat (Leheska et al., 2002; Rosenvold & Andersen, 2003). However, the molecular 404

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tropomyosin, myosin binding protein C and so on, were identified to be acetylated at multiple locations. In fact, muscle contraction related proteins were the largest cluster of acetylproteins identified in the protein–protein interaction network analysis using total acetylated proteins (data not shown). Second, a network of muscle contraction was identified at 0 and 0.5 h PM using the differentially acetylated proteins between the Control and the Transport or the Rest group. As preslaughter transport and lairage induces stress and muscle contraction in animals, these identified networks reflected that protein lysine acetylation was probably involved in animal stress response and muscle contraction, though it was not clear whether it is a regulator or just a result of muscle contraction. Third, most differentially acetylated proteins in the identified networks at 1 h PM were up-regulated in the rested muscle. As lairage accelerates the onset of rigor mortis and increases muscle tension at 3 h PM in pork muscle (Dokmanovic et al., 2015), the up-regulated acetylproteins in the Rest group at 1 h PM were probably related to the earlier onset of rigor mortis and/or increased muscle tension of the Rest group at this time point. In addition, most acetylproteins in the networks were up-regulated in the stressed muscle at 1 and 4 h PM when compared to the control. As the onset of rigor mortis is determined by the depletion of ATP in muscle and preslaughter stress decreases ATP in muscle at 1 and 4 h PM (Shen, Means, Thompson, et al., 2006), it is logical that the onset of rigor mortis took place earlier in the Transport and Rest groups compared to the control and more acetylprotein were up-regulated at 1 h PM (Rest) or at 1 and 4 h PM (Transport). Correspondingly, higher shear force was determined in the Transport and Rest groups compared to the control. These results suggest that protein lysine acetylation is involved in the onset of rigor mortis and may impact meat tenderness. Although some literature reports that acetylation of myosin heavy chains regulates its ATPase activity and sliding velocity (Samant, Pillai, Sundaresan, Shroff, & Gupta, 2015) and deacetylation of cortactin promotes muscle contraction (Li et al., 2014), the role of lysine acetylation in muscle contraction and even muscle development is far from well understood.

rate-limiting enzymes in glycogenolysis/glycolysis is condition-dependent. Although it is well believed that protein lysine acetylation regulates metabolism, but the regulation mechanisms of most metabolic enzymes by acetylation remain unclear. Glycogen phosphorylase (GP) is an acetylprotein. Acetylation of GP at K470 promotes its dephosphorylation and inactivation in human liver (Zhang et al., 2012). In the present study, GP was not detected to be acetylated at K470, but the acetylation of the brain form GP (PYGB) at K505 was down-regulated in both the stressed and rested muscle at 0.5 h PM, when compared to the control. As protein lysine acetylation is not conserved across tissues (Lundby et al., 2012), it is likely that the acetylation of GP is different in human liver and porcine skeletal muscle. The acetylation of PYGB at K505 may correspond to the acetylation of GP at K470 in human liver; thus the deacetylation of PYGB at K505 may increase glycogenolysis in PM muscle in response to preslaughter transport and extended lairage time. Pyruvate kinase catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating pyruvate and ATP. A previous study reports that acetylation of the M2 isoform of pyruvate kinase (PKM2) at K305 decreases its enzymatic activity and promotes lysosomal degradation (Lv et al., 2011). Deacetylation of this site by SIRT2 promotes PKM2 tetramer formation and increases its enzymatic activity (Park et al., 2016). Ten lysine residues (K171, K160, K320, K484, K507, K215, K144, K233, K98, and K195) of pyruvate kinase (PKM) were detected to be acetylated in the present study, of which five (K320, K215, K144, K233, and K98) were down-regulated in the stressed muscle at 0.5 h PM when compared with the control, suggesting that deacetylation of these lysine residues is possibly involved in regulating this enzyme and postmortem glycolysis, as higher pyruvate kinase activity was detected in PM muscle from transported pigs and muscle that became PSE meat (Shen, Means, Thompson, et al., 2006; Shen, Means, Underwood, et al., 2006). Several proteomic surveys (Shakespear et al., 2018; Wang et al., 2010; Zhao et al., 2010) have also reported the acetylation of phosphoglucomutase, phosphoglucose isomerase, enolase, and pyruvate dehydrogenase, but the function of acetylation of these enzymes is not clear. As acetylation frequently inhibits enzymes involved in metabolism (Bontemps-Gallo et al., 2018; Nakayasu et al., 2017; Ryder et al., 2015), the down-regulation of lysine acetylation of these enzymes (PGM1, GPI, ENO1, and DLD) determined in the present study in the muscle from transported pigs early PM may be a mechanism by which preslaughter stress up-regulates glycolysis in PM muscle. In summary, these data fully confirm our hypothesis and previous study that protein lysine acetylation/deacetylation plays an important regulatory role in postmortem glycolysis and some classically non-ratelimiting enzymes, which include PYGB, PGM1, GPI and enolase, could become rate-limiting in postmortem muscle in response to preslaughter stress. In addition, the data propose that deacetylation is likely to be a common way to activate glycolytic enzymes in postmortem muscle and the identified differentially acetylated lysine residues are possibly the mechanisms that preslaughter handling regulated postmortem glycolysis which need to be further defined.

4.4. Pre-slaughter handling, stress response and apoptosis in PM muscle Programmed cell death or apoptosis prior to rigor mortis regulates proteolysis and meat tenderness (Kemp, Sensky, Bardsley, Buttery, & Parr, 2010; Ouali et al., 2006). GO analysis based on biological process revealed that acetylproteins identified in the present study were enriched in cell death. A network of PTPC components, including voltagedependent anion-selective channel protein 1 and 2 (VDAC1 and VDAC2), solute carrier family 25 member 4 and 5 (SLC25A4 and SLC25A5), or ADP/ATP translocase 3 (SLC25A6), was identified for Transport vs. Control and Rest vs. Control at 1 h PM. All the five proteins were up-regulated in acetylation in the Transport or Rest group. As PTPC controls the release of mitochondrial products and regulates mitochondrion initiated apoptosis, it is logical to deduce that preslaughter transport and lairage regulated apoptosis in PM muscle by regulating PTPC function via protein lysine acetylation. Heat shock proteins (HSPs) regulate stress resistance and apoptosis (Takayama, Reed, & Homma, 2003). Some studies report that HSPs may regulate apoptosis, proteolysis and meat tenderness (Kemp & Parr, 2012). In the present study, two HSPs, heat shock 70 kDa protein 1A (HSPA1A) and stress-70 protein (HSPA9) were deacetylated in the Transport and Rest groups at 0 h PM compared to the control. Surprisingly, the acetylation levels of these two proteins were not different between treatments afterwards in the following conversion of muscle to meat, suggesting that acetylation of HSPs may not mediate the effect of preslaughter stress on meat quality development.

4.3. Pre-slaughter handling, rigor mortis and meat tenderness Rigor mortis is basically an event of muscle contraction in the conversion of muscle to meat. Although the function of protein lysine acetylation in muscle contraction is fundamentally unclear, as expected, the dynamic and differential acetylation of contractile and noncontractile proteins (such as ATP production and calcium signaling related acetylproteins) detected in the present study suggests that lysine acetylation is probably involved in muscle contraction and postmortem rigor mortis. First, the highest number of acetylation sites was identified in myosin heavy chains, with 80, 44, and 38 acetylation sites assigned to MHC-2x, MHC-β and MHC-2b, respectively, in the present study. In addition, many myofibrillar proteins, including actin,

4.5. Other events involving protein lysine acetylation in porcine muscle Previous studies report that mitochondrial proteins are commonly acetylated (Ali et al., 2018). As reported in the literature, mitochondrial 405

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acetylproteins were found to be enriched in porcine muscle by GO analysis in the present study, including all enzymes in TCA cycle and many proteins related to fatty acid metabolism, amino acid metabolism, electron transport and oxidative phosphorylation. In addition, many acetylproteins related to aerobic respiration were differentially expressed between treatments at very early PM stage (0–0.5 h PM). A network of TCA cycle was identified for Transport vs. Control and Rest vs. Control at 0 h PM (Fig. 5). A small network of electron transport chains was identified for Rest vs. Transport at 0 and 0.5 h PM. These data indicated that protein lysine acetylation regulated aerobic metabolism in muscle in response to stress. A network of TCA cycle with all acetylproteins in it up-regulated was identified at 1 or 4 h PM for the three comparisons between treatments (Fig. 5). It is not clear why all these acetylproteins were up-regulated in the network, as oxygen in muscle should be depleted and aerobic metabolism stopped at this stage. The up-regulation of these acetylproteins could be an indicator of the onset of rigor mortis and a result of uncontrolled non-enzymatic acetylation after cell death. Calcium is important to meat tenderness as it acts as an activator of muscle contraction and calpain, the protease believed to be primarily responsible for PM proteolysis and meat tenderization. In the present study, acetylproteins were found to be enriched in the calcium signaling pathway by KEGG analysis. Several proteins involved in calcium release from and uptake to sarcoplasmic reticulum (SR) were differentially acetylated between the three treatments (Table S5). Calsequestrin 1 (CASQ1), a calcium-binding protein and Ca2+ store in SR, decreased in acetylation in the stressed and rested muscle compared to the control at 0 h PM, but increased in acetylation in the rested muscle at 1 h PM compared to both the control and stressed muscle. Ryanodine receptor 1 (RYR1), a calcium release channel of SR, was up-regulated in acetylation in the stressed muscle compared to the control at 0.5 h PM. Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1), a Ca2+ ATPase responsible for the reuptake of cytosolic Ca2+ into the SR, was deacetylated in the rested muscle at 0 h PM when compared to the control. The differential acetylation of these proteins suggests that preslaughter handling regulates calcium signaling in PM muscle and probably meat quality.

This work was supported by the National Natural Science Foundation of China (31571862) and National Top Disciplines Development Project for Innovation Teams (kxk201801004). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.04.122. References Ali, I., Conrad, R. J., Verdin, E., & Ott, M. (2018). Lysine acetylation goes global: from epigenetics to metabolism and therapeutics. Chemical Reviews, 118(3), 1216–1252. Bontemps-Gallo, S., Gaviard, C., Richards, C. L., Kentache, T., Raffel, S. J., Lawrence, K. A., ... Gherardini, F. C. (2018). Global profiling of lysine acetylation in borrelia burgdorferi B31 reveals its role in central metabolism. Frontiers in Microbiology, 9, 2036. Chen, Z., Luo, L., Chen, R., Hu, H., Pan, Y., Jiang, H., ... Gong, Y. (2018). Acetylome profiling reveals extensive lysine acetylation of the fatty acid metabolism pathway in the diatom phaeodactylum tricornutum. 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5. Conclusion Protein lysine acetylation was comparatively profiled in PM muscle of pigs with or without preslaughter stress and resting. Totally 771 acetylpeptides containing 681 lysine acetylation sites mapping to 176 acetylproteins were identified in PM porcine muscle. These acetylproteins were enriched in muscle contraction, carbohydrate metabolism, cell apoptosis and calcium signaling that are important to the PM conversion of muscle to meat. Preslaughter stress and resting had a complicated influence on the dynamic protein lysine acetylation/deacetylation and altered acetylome in PM muscle in an acetylation site and PM time-dependent manner. Preslaughter handling may be associated with glycolysis in PM muscle and the overall meat quality via acetylation/deacetylation of multiple enzymes related to glycogenolysis/glycolysis at early PM stage, regulate muscle contraction, rigor mortis and meat tenderness via acetylation/deacetylation of many contractile and non-contractile, ATP production and calcium signalingrelated proteins, and finally regulate animal stress response, cell apoptosis in PM muscle and meat tenderization via acetylation/deacetylation of HSPs and PTPC component proteins. This study provides the first overview of acetylome in PM muscle as affected by preslaughter handling and broadened our knowledge of the biochemistry regulating PM meat quality development. Declaration of Competing Interest None. 406

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