Isobaric Tags for Relative and Absolute Quantification (iTRAQ

Salbutamol, a selective β2-agonist, endangers the safety of animal products as a result of illegal use in food animals. In this study, an iTRAQ-based...
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iTRAQ-based untargeted quantitative proteomic approach to identify change of the plasma proteins by salbutamol abuse in beef cattle Kai Zhang, Chaohua Tang, Xiaowei Liang, Qingyu Zhao, and Junmin Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04397 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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Journal of Agricultural and Food Chemistry

iTRAQ-based untargeted quantitative proteomic approach to identify change of the plasma proteins by salbutamol abuse in beef cattle Kai Zhang,†,‡ Chaohua Tang,†,‡ Xiaowei Liang,†,‡ Qingyu Zhao,†,‡ Junmin Zhang†,‡,* †

State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese

Academy of Agricultural Sciences, Beijing 100193, China ‡

Scientific Observing and Experiment Station of Animal Genetic Resources and

Nutrition in North China, Ministry of Agriculture, Beijing 100125, China *

Corresponding author, E-mail: [email protected]; Fax: 86-10-62815537; Tel:

86-10-62815852.

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ABSTRACT

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Salbutamol, a selective β2-agonist, endangers the safety of animal products because of

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illegal use in food animals. In this study, an iTRAQ-based untargeted quantitative

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proteomic approach was applied to screen potential protein biomarkers in plasma of

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cattle before and after treatment with salbutamol for 21 days. A total of 62 plasma

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proteins were significantly affected by salbutamol treatment, which can be used as

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potential biomarkers to screen for the illegal use of salbutamol in beef cattle.

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Enzyme-linked immunosorbent assay measurements of five selected proteins

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demonstrated the reliability of iTRAQ-based proteomics in screening of candidate

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biomarkers among the plasma proteins. The plasma samples collected before and after

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salbutamol treatment were well separated by principal complement analysis (PCA)

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using the differentially expressed proteins. These results suggested that an

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iTRAQ-based untargeted quantitative proteomic strategy combined with PCA pattern

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recognition methods can discriminate differences in plasma protein profiles collected

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before and after salbutamol treatment.

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KEYWORDS: salbutamol, cattle, plasma, iTRAQ proteome, biomarkers, PCA

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INTRODUCTION

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β2-Agonists, are traditionally used for therapeutic purposes in humans and as well as

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bronchodilator and tocolytic agents in veterinary medicine,1,2 but are also illegally used

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for anabolic purposes to improve animal growth and feed efficiency by reducing fat

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deposition and increasing protein synthesis and carcass leanness.3,4 Because the residues

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of these compounds in animal tissues could pose potential hazards to human health,5–7

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China,8 the European Union,9 and most other countries have prohibited the use of

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β2-agonists as growth promoters in animal husbandry. However, β2-agonists, like

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salbutamol, are repeatedly discovered during routine control by national authorities.10

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To ensure the safety of animal products and monitor illegal usage, many rapid and

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sensitive analytical methods have been developed for analysis of β2-agonist residues in

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various food matrices. These methods, such as enzyme immunoassays and gas or liquid

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chromatography (LC) coupled to mass spectrometry (MS),11–14 are all based on existing

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drugs for the detection of residues in animal tissues. To escape from residue detection

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during routine control, β2-agonists like salbutamol are eliminated rapidly and usually

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below the limit of detection within several days.15 Several unknown β2-agonist

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analogues used illicitly as growth promoters in animal production have been

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discovered.16–18 In addition, the use of low dose “cocktails” that exert a synergetic effect

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and exhibit similar growth promotion characteristics has been reported in food

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animals.19 The use of both unknown β2-agonists and low dose “cocktails” render routine

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screening approaches difficult to implement. In this context, strategies based on the

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detection of physiological actions of anabolic practices are promising approaches to 3

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screen for the illegal use of β2-agonist.20 Over the past few years, omics-based

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technologies, such as transcriptomics, proteomics, and metabolomics, have emerged as

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untargeted global strategies to highlight candidate-biomarkers to tackle illegal practices.

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Several reports have described the use of non-targeted metabolomics approaches to

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screen for the abuse of β2-agonists, like salbutamol, in food animals and athletes.21–24

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Some papers have described the use of comparative two-dimensional gel electrophoresis

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(2DE) proteomics to identify plasma proteins as markers of growth promoter abuse in

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cattle.25,26 However, to the author’s knowledge, the use of proteomics-based strategies to

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screen for the abuse of β2-agonists, such as salbutamol, in food animal has not been

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extensively reported except for clenbuterol.27 Isobaric tags for relative and absolute

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quantification (iTRAQ) is a common labeling strategy in proteomics that has been

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increasing used in the past few years. iTRAQ-based proteomic technologies have been

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proposed as more accurate and reliable methods for the quantitation of proteins than

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traditional 2DE proteomic analysis.28

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Therefore, an iTRAQ-based untargeted quantitative proteomics approach was applied

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to plasma samples collected from cattle before and after salbutamol treatment for 21

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consecutive days to identify differently expressed proteins as potential biomarkers to

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screen for the abuse of salbutamol in beef cattle.

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MATERIALS AND METHODS

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Chemicals and Reagents

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Salbutamol hydrochloride (95% purity) was provided by the Institute of Quality

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Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural 4

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Sciences (Beijing, China). ProteoMiner Protein Enrichment Kits and Bradford protein

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assays were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The

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iTRAQ reagent-8 plex one assay kit was purchased from AB Sciex Pte. Ltd. (Foster

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City, CA, USA). Dithiothreitol and iodoacetamide were obtained from Promega Co.,

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Ltd. (Madison, WI, USA). Acetone, tetraethylammonium bromide (TEAB), sodium

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dodecyl sulfate (SDS), trypsin, monobasic potassium phosphate (KH2PO4), phosphoric

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acid (H2PO4), potassium chloride (KCL), and isopropanol were purchased from

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Sigma-Aldrich (Schnelldorf, Switzerland). Formic acid and acetonitrile were obtained

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from Fisher Scientific (Loughborough, UK). All solvents used in this study were

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analytical grade or higher.

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Experimental Animals

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In this experiment, four seven-month-old male Chinese Simmental beef cattle weighting

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281.11 ± 29.36 kg (mean ± standard error) were housed in a single stall barn for

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tie-down feeding with ad libitum access to feed and water. The cattle were orally

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administrated salbutamol at a dose of 0.15 mg/kg body weight/d for 21 consecutive

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days. Blood samples were collected before and after treatment for 21 days from the

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jugular vein into heparinized tubes, centrifuged at 1450 × g for 10 min to obtain plasma

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samples, which were then stored at −80 °C. The study protocol was approved by the

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Animal Welfare Committee of the Institutes of Animal Science, Chinese Academy of

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Agricultural Sciences.

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Sample Preparation

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Plasma samples were defrosted and centrifuged at 10000 × g for 10 min. Then, 5

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ProteoMiner™ Protein Enrichment kits (Bio-Rad Laboratories, Inc.) were used to

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simultaneously dilute high-abundance proteins and concentrate low-abundance proteins,

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as described by the manufacturer’s instructions, and subsequently, reduced using 10

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mM dithiothreitol, in a 56 °C water bath for 1 h. Then, iodoacetamide was quickly

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added to a final concentration of 55 mM, under darkness for 1 h. Afterward, 4 × volume

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of cold acetone was added and the samples, which were stored at −20 °C for 3 h and

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then centrifuged at 20000 × g (4 °C) for 20 min. The precipitants were collected, mixed

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with 300 µL of buffer (50% TEAB and 0.1% SDS), and then sonicated for 3 min.

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Finally, the total protein concentrations were determined using the Bradford method,

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according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc.).

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Digestion and iTRAQ Labeling

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One hundred micrograms of protein from each plasma sample (consistent volume

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containing 0.1% SDS of TEAB) were combined with 3.3 µg of trypsin (1 µg/µL) in a

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37 °C water bath for 24 h. Afterward, 1 µg of trypsin was added to the sample, which

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was then incubated in a 37 °C water bath for 12 h, freeze-dried in digestion fluid and

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redissolved in 30 µL of TEAB (water:TEAB, 1:1, by volume). The iTRAQ reagents

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were allowed to adjust to room temperature, then added to 70 µL of isopropanol and

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vortexed for 1 min. The resulting peptides were labeled using with iTRAQ reagents for

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2 h at room temperature. Then, the eight labeled peptides were pooled together. The four

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plasma samples collected before treatment were labeled with iTRAQ reagents 113, 114,

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115, and 116, while the four treatment plasma samples were labeled with iTRAQ tags

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117, 118, 119, and 121, respectively. 6

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Strong Cationic-Exchange Chromatography Separation

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The combined peptide mixtures were dried in a Speedvac vacuum concentrator and

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dissolved in 1 µL of buffer A (10 mM KH2PO4 in 25% acetonitrile). After adjusting the

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pH to 3.0 with H2PO4, the sample was subjected to strong cationic-exchange

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chromatography (SCX) using a silica-based column (250 × 4.6 mm, Phenomenex, Inc.,

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Torrance, CA, USA). A total of 16 fractions were collected with a gradients of buffer B

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(10 mM KH2PO4 and 2 M KCL in 25% acetonitrile, pH 3.0) as follows: 0% B for 45

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min, 0–5% B for 1 min, 5%–30% B for 20 min, 30%–50% B for 5 min, 50% B for 5

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min, 50%–100% B for 5 min, and 100% B for 5 min. The fractions were desalted with a

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strata-X C18 column (Phenomenex, Inc.).

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LC-MS/MS Analysis and Protein Quantification

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For chromatographic separation, a Dionex ultimate 3000 NanoLC system (Dionex,

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Sunnyvale, CA, USA) was used. Separation was carried out on a reversed phase C18

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column (100 × 75 mm, 5 µm, 300 Å, Agent Technologies, Santa Clara, CA, USA).

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Solvent A was composed of 0.1% formic acid in water and solvent B was composed of

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0.1% formic acid in acetonitrile. Peptides were eluted at a flow rate of 400 nL/min with

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the flowing gradient conditions: 5% B for 10 min, 5%–30% B for 30 min, 30%–60% B

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for 5 min, 60%–80% B for 3 min, 80% B for 7 min, 80%–5% B for 3 min, and 5% B for

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7 min. The elutants were directly subjected to Q-Exactive MS (Thermo Fisher Scientific,

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Waltham, MA, USA), setting in positive ion mode and data-dependent manner with full

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MS scan range from 350 to 2000 Da, full scan resolution of 70000, MS/MS scan

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resolution of 17500, capillary temperature of 320 °C, ion source voltage of 1800 V, 7

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MS/MS scan with minimum signal threshold of IE+5, and an isolation width of 2 Da.

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To evaluate the performance of this MS on the iTRAQ-labeled samples, two MS/MS

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acquisition models, and higher collision energy dissociation were employed.

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Protein Identification and Quantification

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For protein identification, the raw files were processed using Proteome Discover 1.3

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software (Thermo Fisher Scientific) and searched with Mascot 2.3.0 (Matrix Science,

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London, UK) against the bovine genome with an in-house Uniprot database (02-2016,

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32352 entries). Carbamidomethyl (C) was set as a fixed modification, while oxidation

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(M), Gln→Pyro-Glu (N-term Q), iTRAQ 8 plex (K), iTRAQ 8 plex (Y), and iTRAQ 8

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plex (N-term) were set as variable modifications. The peptide tolerance was set to 15

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ppm, and the MS/MS tolerance was set to 20 mmu. Trypsin was selected as the enzyme

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and up to one missed cleavage was allowed. All reported data were based on 99%

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confidence for identification of proteins and peptides as determined by a false discovery

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rate of ≤ 1%. Each confident protein identification involved at least one unique peptide.

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The quantitative protein ratios were weighted and normalized by the median ratio in

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Mascot. Only protein identification that was inferred from the unique peptide

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identification in all four biological replicates was included for subsequent analysis.

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Statistical analysis was performed using a one-way analysis of variance. A probability

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(P) value of < 0.05 and fold change > 1.2 or < 0.83 (as previous study defined)29 were

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considered to indicate a differentially expressed protein.

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Enzyme-linked Immunosorbent Assay (ELISA) Validation

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To confirm the iTRAQ-based proteomic results, concentrations of five selected proteins 8

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in each cattle plasma sample were measured using a bovine ELISA kit (Nanjing

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Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China), according to the

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manufacturer's instruction, respectively. Briefly, 50 µL of a diluted standard or plasma

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sample was added into the wells of 96-well plates, which were combined with 50 µL of

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biotinylated working solution and incubated at 37 °C for 60 min. Then, the plates were

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washed five times with washing solution. Afterward, 50 µL of horseradish peroxidase

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working solution were added to each well and the plate was incubated at 37 °C for 60

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min. After repeating the plate-washing procedure, 50 µL of chromogenic solutions A

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and B were added to each well and then plate was incubated at 37 °C for 10 min away

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from light. Afterward, 50 µL of stop solution were added to each well and the optical

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density at a wavelength of 450 nm was measured using the Infinite M200 Microplate

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Reader (Tecan Group Ltd., Männedorf, Switzerland).

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Data Analysis

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Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)

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functional analyses of differentially abundant proteins were performed using the

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DAVID online software tool (https://david.ncifcrf.gov/summary.jsp). The GO project

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categorized all of the differentially expressed proteins into three groups: cellular

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components, molecular functions, and biological processes. Significant GO terms and

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pathways with P < 0.05 are presented. The identified differentially expressed proteins

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were analyzed using the PCA program included with the SIMCA 13.0 software package

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(MKS Umetrics AB, Umea, Sweden). Statistical analysis was performed using IBM

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SPSS Statistics for windows, version 20.0 (IBM-SPSS, Inc., Chicago, IL, USA). The 9

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Student's t-test was employed to identify significant differences in protein

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concentrations before and after salbutamol treatment. A P-value < 0.05 was considered

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statistically significant.

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RESULTS

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Identified Proteins and Function Analysis

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The workflow of the iTRAQ-based proteomic approach for comparison of the cattle

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plasma proteome before and after salbutamol treatment is shown in Figure 1. In total,

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472 proteins were identified from Chinese Simmental beef cattle before and after

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salbutamol treatment for 21 consecutive days (Supporting Information, Table S1). Of

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these, 441 of the identified proteins were quantified by the iTRAQ-based proteomic

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approach and each group included four biological replicates (Supporting Information,

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Table S2). The labelling efficiency based on peptide was 98%. Statistical analysis of the

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coefficient of variation of the four biological in this work was below 20%. A total of 62

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significant differentially expressed proteins were detected by the analysis of variance

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test (P < 0.05) and conformed to a fold change of >1.2 or