Proteome Analysis Using Isobaric Tags for Relative and Absolute

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Proteome analysis using iTRAQ reveals the alterations in stress-induced dysfunctional chicken muscle Tong Xing, Chong Wang, Xue Zhao, Chen Dai, Guanghong Zhou, and Xinglian Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05835 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Proteome analysis using iTRAQ reveals the alterations in

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stress-induced dysfunctional chicken muscle

3 Tong Xing,† Chong Wang,† Xue Zhao,† Chen Dai,‡ Guanghong Zhou† and Xinglian Xu*,†

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Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Synergetic

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Innovation Center of Food Safety and Nutrition, College of Food Science and Technology,

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Nanjing Agricultural University, Nanjing 210095, China

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Experimental Teaching Center of Life Science, Nanjing Agricultural University, Nanjing 210095,

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People’s Republic of China

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* Corresponding author: Xinglian Xu. Email: [email protected]; Tel.:+86-25-84395939; Fax:

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+86-25-84395939.

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ABSTRACT: The current study was designed to investigate the changes in the protein profiles of

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pale, soft and exudative (PSE)-like muscles of broilers subjected to transportation under high

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temperature conditions, using isobaric tags for relative and absolute analysis quantitation (iTRAQ).

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Arbor Acres chickens (n = 112) were randomly divided to two treatments: unstressed control

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(CON) and 0.5 h of transport (T). Birds were transported based on a designed protocol. Pectoralis

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major (PM) muscle samples in the T group were collected and classified as normal (T-NOR) or

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PSE-like (T-PSE). Plasma activities of stress indicators, muscle microstructure and proteome were

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measured. Results indicated that broilers in the T-PSE group exhibited higher activities of plasma

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stress indicators. The microstructure of T-PSE group showed a looser network and larger

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intercellular spaces in comparison to the other groups. Proteomic analysis, based on iTRAQ

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revealed 29 differentially expressed proteins in the T-NOR and T-PSE groups that were involved

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in protein turnover, signal transduction, stress and defense, calcium handling, cell structure and

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metabolism. In particular, proteins relating to the glycolysis pathway, calcium signaling, and

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molecular chaperones exhibited significant differences that may contribute to the inferior

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postmortem meat quality. Overall, the proteomic results provide a further understanding about the

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mechanism of meat quality changes in response to stress.

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KEYWORDS: Broiler; Stress; Proteome; Glycolysis; Calcium signaling; Molecular chaperone

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INTRODUCTION

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During the past few decades, the selection of broilers for augmented growth rate and breast

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yield has been accompanied with severe muscle dysfunction and pectoral myopathy, among which

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pale, soft and exudative (PSE)-like syndrome is the most serious 1. Due to the abnormal color,

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lower water holding capacity (WHC), and impaired functional properties, it has limited value in

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the meat-processing sector, thereby causing huge losses to the modern poultry industry 2. Previous

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studies indicated that pre-slaughter stressors, such as transportation conditions and environmental

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heat, have deleterious effects on broiler welfare, and may increase the incidence of PSE-like meat

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3, 4

.

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The emergence of novel proteomic techniques in recent years has greatly aided with

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developed understanding of biological mechanisms and the discovery of different meat quality

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biomarkers

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stress and early postmortem chilling processes, which are attributed to increased postmortem

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glycolysis and a rapid decrease of pH 7. The use of two-dimensional electrophoresis, combined

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with MALDI-TOF-MS, indicated that myosin-light chain 1, troponin T, and alpha-crystalline were

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less proteolyzed and HSP27 was undetectable in the PSE zones of pig semimembranosus muscle,

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indicating a probable difference in the underlying physiological mechanism derived from stress 8.

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Di Luca et al.

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and found proteins differ in metabolism, oxidative stress response, muscle structure, and signal

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transduction. The decreased expression of stress-related proteins was correlated with an impaired

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WHC when compared with the normal, illustrating the significance of the stress response in the

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improvement of WHC.

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. Factors that directly cause the incidence of PSE-like meat are genetic, slaughter

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compared the protein profiles of pork with high and low drip loss phenotypes,

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Research on alterations in proteins in pectoralis major (PM) muscle of broilers in response to

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stress and the mechanism triggering the development of PSE-like meat is limited. In addition, the

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reason for the variation in meat quality caused by intra-broiler variability in response to stress

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remains unclear. To depict the potential mechanism of meat quality development, isobaric tags for

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relative and absolute quantitation (iTRAQ)-labeling combined with LC-MS/MS were used for

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identifying the changes in protein profiles in the PM muscle of broilers, based on categorized

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normal and PSE-like muscles collected after subjecting broilers to acute transport under high

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ambient temperature.

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MATERIALS AND METHODS

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Experimental design. Arbor Acres broiler chickens (n = 112) at 42 day of age, with an

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average weight of 2.75 kg, were divided to two treatments: control (CON) and 0.5 h transport (T).

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All procedures were consistent as recommended by the Commercial Broiler Management Guide.

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Feed and water were withdrawn 8 h prior to slaughter. Birds were held in crates (760mm ×

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480mm × 380mm) containing seven birds each with 8 replicates, totaling 56 birds that were then

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distributed at the rear of a truck. The birds in the T group were transported at an average speed of

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45 km/h on a flat road. The ambient temperature was about 33°C, and the temperature inside the

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crate was about 41°C during transportation. All birds were slaughtered in accordance with the

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commercial slaughter process, using electrical stunning, then exsanguination via the carotid

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arteries and jugular veins, followed by scalding and evisceration. Blood was collected in

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heparinized tubes and mixed gently. The tubes were centrifuged at 2,000 g at 4°C for 10 min and

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the supernatant was kept at -20°C for stress indicator determinations. Immediately after

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exsanguination, ten grams of the right PM muscle was taken quickly, frozen in liquid nitrogen and 4

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stored at -80°C for proteome measurements. The other muscles were used for assessment of

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muscle pH at 0.5 h, meat quality and histological analysis at 24 h postmortem.

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Sample collection. The criteria for categorization of muscles in the T group as normal (46
1.5 or