Comparative analysis of whey proteins in donkey colostrum and mature milk using quantitative proteomics

Food Research International 127 (2020) 108741

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Comparative analysis of whey proteins in donkey colostrum and mature milk using quantitative proteomics Weixuan Li, Mohan Li, Xueyan Cao, Hongjiao Han, Fanhua Kong, Xiqing Yue

T

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College of Food Science, Shenyang Agricultural University, Shenyang, Liaoning Province, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Donkey milk Colostrum Mature milk Proteomic Parallel reaction monitoring Biological functions

Donkey milk is attracting increasing attention as a nutritional milk source similar to human milk. In this study, we carried out qualitative and quantitative analysis of the donkey whey proteome using a label-free proteomic approach, combined with parallel reaction monitoring (PRM) as a validation method. A total of 300 whey proteins were identified in donkey colostrum (DC) and donkey mature (DM) milk, of which 18 were differentially expressed (P < 0.05) between the two types of milk. Gene ontology (GO) analysis showed that differentially and uniquely expressed proteins were mainly involved in cellular processes, response to stimulus, metabolic processes, and biological regulation. Their molecular functions included binding, catalytic activity, and molecular functional regulation, and their main annotated areas of origin were the cell, cell-part, and the extracellular region. Most differentially and uniquely expressed proteins were linked with malaria, systemic lupus erythematosus, or antigen processing and presentation. Our results provide insight into the complexity of the donkey whey proteome and molecular evidence for nutritional differences between different lactation stages.

1. Introduction Milk is a natural fluid produced by mammalian mammary glands and provides perfect nutrition for newborns. Its benefits as a food for infants are linked to its bioactive constituents, particularly proteins. Many functional proteins in milk, such as immunoglobulins, lactoferrin, and α-lactalbumin, can help infants to establish their immune systems and prevent infectious diseases postpartum. It is well known that breast milk is the best source of nutritional milk for infants and young children. However, infants cannot always be fully nourished with breast feeding. Some babies need formula milk powder for nutrition. Milk is the main raw material of infant formula milk powder, and is also the main cause of protein allergy in children younger than three years (Martini, Altomonte, Manica, & Salari, 2015). Therefore, it is critical to analyze milk sources for similarity to human breast milk. Donkey milk has good palatability and low allergenicity, and is now considered to be a potential substitute for human milk (Gubić et al., 2015). Its lactose and protein contents are close to those of human milk (Contarini, Pelizzola, Scurati, & Povolo, 2017). Studies by Cunsolo et al. show that the low allergenic properties of donkey milk with respect to cow one seem to be related to the low total protein content, the low ratio of caseins to whey fraction (Cunsolo et al., 2017). Donkey milk has

attracted more attention in recent times due to its bioactive constituents, and it is now a significant subject of research on childhood nutrition. Studies with animal models have found that the bioactive components of donkey milk can improve immune response, and show anti-inflammatory effects (Šarić et al., 2014; Souroullas, Aspri, & Papademas, 2018; Yvon et al., 2018). Animal model experiments have also shown that donkey milk has the benefits of reducing blood lipids, controlling body weight, and improving energy metabolism (Martini, Altomonte, Licitra, & Salari, 2018; Ragona et al., 2016). Another study reported that it contains higher levels of lysozyme than human milk (Elhatmi et al., 2015). High levels of this protective antimicrobial factor are beneficial to the intestinal health of people with low immunity, and it has an inhibitory effect on the proliferation of tumor cells in vitro (Yvon et al., 2018). Analysis of the nutritional components in donkey milk could also be relevant to the scientific improvement of dairy formula products designed to replace or supplement human breast milk. Casein, whey protein, and milk fat globule membrane protein are the major proteins in milk (Cao et al., 2017; Murgia et al., 2016). In the past, whey was treated as production waste during the cheese production process, and its nutritional value was ignored (Giroux, Veillette, & Britten, 2018). Whey protein accounts for about 20% of the total protein content in cow milk, which is mainly composed of β-

Abbreviations: GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PRM, parallel reaction monitoring; IT, isolation time; HCD, higher-energy collisional dissociation; AGC, automatic gain control; BCA, bicinchoninic acid assay; UT buffer, 8 M Urea and 150 mM Tris-HCl, pH 8.0 ⁎ Corresponding author. E-mail address: [email protected] (X. Yue). https://doi.org/10.1016/j.foodres.2019.108741 Received 9 June 2019; Received in revised form 30 September 2019; Accepted 2 October 2019 Available online 14 October 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved.

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lactoglobulin (β-LG), and α-lactalbumin (α-LA) (El-hatmi et al., 2015; Li, Ding, Wan, Liu, & Du, 2014; Medeiros et al., 2018). The reliability of using proteomic techniques to analyze the interspecific differences in whey protein has been established (Roncada, Stipetic, Bonizzi, Burchmore, & Kennedy, 2013), and previous studies have analyzed the proteomes of different species. For example, quantitative differences in whey proteins among Murrah, Nili-Ravi, and Mediterranean buffaloes were studied by a tandem mass tag (TMT) proteomic approach (Li et al., 2018). The whey proteomes of human, camel, donkey, goat, and cow were analyzed by anion-exchange fast protein liquid chromatography (FPLC) combined with polyacrylamide gel electrophoresis (El-hatmi et al., 2015). In addition, the differences among whey proteomes from different lactation stages have also been analyzed by some studies. Human and bovine milk whey proteins from different lactation stages have been characterized using an isobaric tag for relative and absolute quantification (iTRAQ) (Yang et al., 2017). Other studies used quantitative and qualitative analyses including lectin enrichment combined with liquid chromatography tandem mass spectrometry to compare whey proteins and whey N-glycoproteins from human and bovine milk during different lactation stages (Cao et al., 2017, 2019). Highly enriched whey proteins in human colostrum include Ig kappa chain V-III region IARC/BL41, Ig kappa chain V-I region EU, chitinase-3-like protein 2, myosin-reactive immunoglobulin heavy chain variable region, and serum amyloid A protein. These whey proteins are closely related to immune function, especially serum amyloid A protein, which plays an important role in the immune system (Sun & Ye, 2016). Fatty acidbinding protein, which was identified as the major whey protein in human mature milk, has the function of transporting plasma fatty acids (Yang et al., 2017). Ig gamma-1 chain C region and Alpha-lactalbumin are highly expressed proteins in bovine colostrum and bovine milk respectively. They are involved in immune functions and calcium binding, respectively (Yang et al., 2017). Cao et al. showed that the Nglycoproteome from human and bovine colostrum was mainly involved in protein binding function, while the N-glycoproteome from human and bovine mature milk was mainly involved in hydrolase activity function (Cao et al., 2019). The results of these studies indicate that the composition and function of the whey proteome are different not only among species, but also among lactation stages. It is also worth highlighting the fact that, while milk whey is the main component of infant formula milk powder, the compositions of human and bovine whey are not the same. Compared with bovine milk, the whey proteome composition of donkey milk is closer to that of human milk (Zhou, 2010). In previous study, Vincenzo Cunsolo et al. has identified the donkey whey proteome (Cunsolo et al., 2011; Cunsolo, Saletti, Muccilli, & Foti, 2007). However, the donkey whey proteome at different lactation stages has not been comprehensively studied. The comprehensive identification of donkey whey proteins at various lactation stages could therefore provide valuable information for the improvement of the nutritional components of infant formula milk powder. The aim of this study was to characterize the whey proteins in donkey milk, and explore their potential nutritional functions among the different lactation stages (colostrum and mature milk). We used a label-free quantitative proteomic approach. The expression pattern of whey protein in donkey milk at different lactation stages was compared by hierarchical cluster analysis. GO functional annotation, KEGG metabolic pathway, and protein-protein interaction network analyses were used to explore the biological functions and metabolic pathways of donkey whey proteins. Finally, we used PRM technology to verify the abundance of interesting proteins in donkey whey at different lactation stages.

Dalian, China. Thirty donkey colostrum (0–5 days postpartum) and thirty mature milk (15 days to 6 months postpartum) samples were obtained from 30 healthy Dezhou donkeys. All of the 30 donkeys were aged between 2 and 4 years, and all were first-born. They were mainly fed on alfalfa and with weight between 250 and 350 kg. The average daily milk yield of each donkey ranges from 1.5 kg to 2.5 kg. Each sample of donkey milk was collected by automatic suction pump at three separate times over a day and then mixed to eliminate the influence of sampling time point on milk samples. Thirty colostrum and thirty mature milk samples were collected. The samples were placed in dry ice at −20 °C, transported to the laboratory, and stored in a cryogenic refrigerator at −80 °C. The purpose of our study was to compare the donkey whey proteomes from different lactation stages, not between individual donkeys. In order to avoid the influence of individual differences on the whey proteins at different lactation stages, we mixed groups of 10 milk samples before the analysis. This allowed three biological replicates while excluding individual differences. The donkey milk samples were centrifuged at 4 °C for 15 min at 10,000g to remove fat. The pH of the skimmed samples was adjusted to 4.6 by adding 33% acetic acid (30 μL) and standing them at 25 °C for 15 min. The casein was removed by adding 30 μL of 3.3 M sodium acetate to the mixture and centrifuging at room temperature for 30 min at 14,000g. We collected the supernatant containing whey protein and used BCA (P0012; Beyotime) protein assays to measure the whey protein concentration. The collection of milk samples was approved by Shenyang Agricultural University and the Chinese Human Research Ethical Committee, and was conducted in accordance with the Declaration of Helsinki and the Nuremberg Code. All experiments were performed according to Chinese laws and institutional guidelines. 2.2. Trypsin digestion of donkey whey fractions A total of 600 μg of whey protein, and a mixture of dithiothreitol with a concentration of 100 mM, were incubated at 90 °C for 120 min. The sample was cooled to room temperature and mixed with UT buffer (8 M Urea and 150 mM Tris-HCl; pH 8.0). The mixed samples were incubated for 0.5 h in th-e dark and filtered with a 10 kDa ultrafiltration filter. Iodoacetamide and UT buffer were added to the ultrafiltration filter and it was left to stand for 1 h at room temperature in the dark. The sample was centrifuged at 14,000g at 4 °C for 20 min, and then washed three times with UT buffer. The protein was then digested for 18 h by adding trypsin buffer (protease/buffer = 1/8) at 37 °C. This was followed by filtration of the digested sample through an ultrafiltration membrane (10-kDa, Millipore, Bedford, USA) and centrifugation at 15,000g at 4 °C for 15 min. The filtrate containing peptide fragments was collected and desalted using a C18 cartridge. After freeze-drying, the peptide segments were re-dissolved in 40 μL 0.1% formic acid solution. The absorbance of the peptide was measured at 280 nm using a UV spectrophotometer. 2.3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis The whey protein was isolated and identified using a combination of Thermo Fisher EASY-nLC 1000 and Q-Exactive liquid chromatographymass spectrometry (LC-MS). The mobile phase consisted of two solutions. Mobile phase A was 0.2% formic acid and 99.8% water. Mobile phase B was 84% acetonitrile, 0.2% formic acid and 15.8% water. Our LC-MS settings were based on the paper by Cao et al (Cao et al., 2017). The whey protein was loaded onto a nanoViper C18 pre-column (Thermo Scientific Acclaim Pepmap100, 2 cm, 100 µm inner diameter, 5 μm) for separation followed by a C18 analytical column (Thermo scientific EASY column, 10 cm, 75 μm inner diameter, 3 μm, C18-A2) at a flow rate of 300 nL min−1. The mass spectrometry settings were as follows: (1) the source was operated at 2.0 kV; (2) the MS was programmed in data-dependent acquisition mode; (3) the survey scan was

2. Experimental 2.1. Sample collection Samples of donkey milk were collected at Dezhou donkey farm in 2

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ID) with at a flow rate of 250 nL min−1. Solvent A solution was 0.1% formic acid, and solvent B solution was 0.1% acetonitrile formic acid (84% acetonitrile). The chromatographic column was equilibrated with 95% liquid solvent. The gradients of liquid phase separation were 2–5% liquid B for 0 to 1 min, 5–30% liquid B for 1–45 min, 30–40% liquid B for 45–47 min, and 100% liquid B for 47–60 min. The separated samples were analyzed by PRM mass spectrometry with a Q Exactive HF mass spectrometer. The full-scan MS scanning range was 300–1800 m/ z. The mass spectrometry resolution was 60,000 (@m/z 200), the AGC target was 3e6, and the maximum IT was 200 ms. After each full MS scan, 20 PRM scans were collected according to the inclusion list. The isolation window was 1.6 m/z, the MS/MS resolution was 30,000 (@m/ z 200), the AGC target was 3e6, the maximum IT was 120 ms, the MS2 activation type was HCD, and the normalized collision energy was 27. The milk samples were detected by PRM, and the original PRM files were analyzed by the software Skyline 3.5.0 (Brendan et al., 2010). Peptide identity was confirmed by matching PRM data with the MS/MS spectral library. This spectral library was created with the dataset generated by a classical data-dependent acquisition (DDA) approach, and the peptide tandem mass spectrometry library was established using the database from NIST (http://chemdata.nist.gov/). Six product transition ions were selected to match the library. The peptide abundance was calculated by normalization of the total ion current (TIC) collected in each scan, and the peptide abundance was summed to quantify the protein.

at 350–1800 m/z with 70,000 resolving power. We applied 120 min mass spectrometry analysis to chromatographic separation of peptide segments by Q-Exactive MS. A gradient was 0% to 55% (v/v) mobile phase B for 110 min and 55% to 100% (v/v) mobile phase B for 5 min, where it was held at 100% (v/v) mobile phase B for 5 min. The resolution of full-scan mass spectrometry was set at 70,000 at 200 m/z and the Automatic gain control (AGC) target was set at 1e6. With 50 ms as the maximum isolation time (IT), the dynamic exclusion operation of 60 s was carried out. The MS2 activation type was set to higher-energy collisional dissociation (HCD), and the isolation window with a masscharge ratio of 2 m/z was selected. The tandem mass spectrometry had a resolution of 17,500 at 200 m/z. The normalized collision energy was set at 30 eV and the underfill ratio was set at 0.1%. 2.4. Identification and quantitative analysis of whey proteins The data obtained from mass spectrometry were analyzed with the software MaxQuant (version 1.5.3.17). Trypsin was used for cleavage and a maximum of two missed cleavages were allowed. Carbamido methyl (C) was used for fixed modifications and oxidation (M) was used for variable modifications. The full-scan MS and MS/MS analysis allowed 6 ppm and 20 ppm mass tolerance for fragment ions, respectively. The maximum false discovery rate (FDR) was set to 0.01 for protein and peptide. Label-free quantification was carried out in MaxQuant (Luber et al., 2010). Protein abundance was calculated on the basis of the normalized spectral protein intensity (LFQ intensity) (Dozio & Sanchez, 2017). The abundances of each peptide were obtained by integrating peak area. The abundances of identified peptides were normalized to the abundances of each unique peptide. The abundance of proteins was obtained by summing all the unique normalized peptide ion abundances (Brendan et al., 2010; Dozio & Sanchez, 2017).

3. Results 3.1. Identification and quantification of the whey proteome in donkey milk by LC − MS/MS We used a label-free proteomic approach to identify the whey proteins in donkey milk. In our study, 288 whey proteins in DC and 287 whey proteins in DM were identified and quantified by LC-MS/MS. Alpha-lactalbumin, beta-lactoglobulin, Lysozyme C, and Immunoglobulin gamma 1 heavy chain constant region were the major whey proteins in donkey colostrum and mature milk. Donkey colostrum milk shared 275 whey proteins with mature milk. As shown in Table 2, there were 13 and 12 uniquely expressed whey proteins identified in donkey colostrum and mature milk, respectively. The uniquely expressed proteins in colostrum milk included the immunoglobulin lambda light chain variable region, zinc-alpha-2-glycoprotein, and thrombospondin. The uniquely expressed proteins in mature milk included histone H3, myristoylated alanine-rich C-kinase substrate, and transcription factor AEBP1.

2.5. Bioinformatics and multivariate analysis The acquired raw MS/MS data were processed using MaxQuant software (version 1.3.0.5) searching against the established Equus databases downloaded from Uniprot. Trypsin was set as the cleavage enzyme for two missed cleavages. Carbamidomethylation was set as the fixed modification, and oxidation of methionine was set as variable modifications. The filter parameter results were set to Peptide false discovery rate (FDR) ≤0.01. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for donkey whey proteins were carried out using the Database for Annotation, Visualization, and Integrated Discovery version 6.8 webbased tool (https://david.ncifcrf.gov/summary.jsp). Hierarchical clustering was conducted using Cluster 3.0 open source software and the data were visualized using Java TreeView. The protein-protein interaction network was analyzed using STRING software, version 10.5, and visualized using Cytoscape open source software, version 3.5.0.

3.2. Differentially expressed whey proteins in donkey colostrum and mature milk As shown in Table 1 and Fig. 1, among 275 common proteins in whey, 18 were differentially expressed, based on the criteria of a difference of at least two-fold, and P < 0.05. We found that there were eleven upregulated and seven downregulated proteins in colostrum milk. The levels of apolipoprotein B, beta-lactoglobulin II, prothrombin, and amine oxidase were higher in colostrum, while the levels of perilipin, cathepsin B, fatty acid-binding protein, and transforming growth factor beta induced were lower in colostrum than in mature milk.

2.6. Liquid chromatography-parallel reaction monitoring/mass spectrometry (LC-PRM/MS) analysis We developed parallel reaction monitoring (PRM) assays to quantify the five candidate functional proteins in a single LC-PRM-MS run. The information on peptide segments suitable for PRM analysis was imported into Xcalibur software for PRM calibration (Codreanu et al., 2017; Dozio & Sanchez, 2017). Each sample was extracted with 10 μg peptide fragments and detected by incorporating 200 fmol standard peptide (PRTC: TASEFDSAIAQDK.). The mass of peptide sequence (Thermo Scientific; Pierce Retention Time Calibration Mixture) was 1389.6503 Da. The observed mass and hydrophobicity Factor of peptide Sequence were 695.8324 Da and 25.88, respectively. The samples were injected into trap column (2 cm × 75 µm ID) and gradient separation was carried out on a Thermo Scientific EASY column (50 cm × 75 µm

3.3. GO analysis of the differentially expressed whey proteins in donkey colostrum and mature milk In order to compare the biological functions of whey proteins between different lactation stages, 43 differentially expressed whey proteins in colostrum and mature milk were analyzed by GO functional annotation. These whey proteins included 18 differentially expressed proteins and 25 specifically expressed proteins. All of 25 specifically 3

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Table 1 Differentially expressed whey proteins in donkey colostrum and mature Milk. UniPort accession

Description

Coverage (%)

Mol/Weight (kDa)

FCa

p value

Change

F6YCW8 A0A1D9CF79 F7BFJ1 F1MZA0 F6XSF7 F2VYX6 C1L3G3 F7CWC8 F7BSX5 F6Q9S8 Q95M34 F6Q4N3 Q6Y1E1 H9GZS6 F6VB94 P10790 F6YNH6 F6Q9R1

Apolipoprotein B Beta-lactoglobulin II Prothrombin Amine oxidase Uncharacterized protein MHC class I antigen (Fragment) Alphas 2 casein B Amine oxidase Uncharacterized protein Tetraspanin Immunoglobulin gamma 1 heavy chain constant region (Fragment) Neural EGF-like 2 Thymosin beta 4 Uncharacterized protein Transforming growth factor beta induced Fatty acid-binding protein, heart Cathepsin B Perilipin

8 89.6 38.4 13 30.7 5.6 43.8 45.3 21.4 10.7 48.1 14.9 31.8 33.4 39.8 67.7 51.8 20.8

511.24 8.73 70.47 58.19 193.02 40.08 18.36 56.94 42.88 23.32 37.44 91.76 5.10 36.96 72.58 14.78 37.72 45.76

17.15 9.63 4.14 3.76 3.38 3.32 3.30 2.99 2.42 2.31 2.28 0.48 0.42 0.39 0.35 0.31 0.27 0.25

0.0048 0.0258 0.0212 0.0221 0.0018 0.0306 0.0110 0.0357 0.0021 0.0357 0.0036 0.0104 0.0499 0.0222 0.0016 0.0216 0.0390 0.0060

↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓

a

FC = fold change, mean value of peak area obtained from DC group / mean value of peak area obtained from DM group. If the fold-change value was more than 2, the relative content of whey protein in DC was higher than in DM, and if the fold-change value was less than 0.5, the relative content of whey protein in DC was less than in DM.

3.4. KEGG pathway analysis of the differentially expressed whey proteins in donkey colostrum and mature milk

expressed proteins included 13 and 12 uniquely expressed whey proteins identified in donkey colostrum and mature milk, respectively. GO functional annotation is divided into three categories: biological processes, molecular functions, and cellular components. As shown in Fig. 2, the prevalent biological processes with the most proteins were cellular processes, response to stimulus, metabolic processes, and biological regulation. Others were involved in regulation of biological processes, cellular component organization or biogenesis, and immune system processes. Three prominent molecular functions of donkey whey proteins were binding, catalytic activity, and molecular function regulation. Molecular transducer activity and transporter activity were also significantly represented. Whey proteins were highly enriched in cell, cell part, and extracellular region. Other enriched origin categories were organelle, extracellular region part, and membrane.

The differentially expressed whey proteins in donkey colostrum and mature milk were related to 86 KEGG pathways; the first 20 pathways are shown in Fig. 3. Malaria, systemic lupus erythematosus, antigen processing, and presentation were the most prevalent pathways. Tetraspanin, thrombospondin 4, and LDL receptor-related protein 1 were involved in the malaria pathway. Uncharacterized protein (F6XSF7), histone H3, and histone H4 were linked with systemic lupus erythematosus. MHC class I antigen, cathepsin B, and MHC class II-associated invariant chain were involved in antigen processing and presentation. The other major pathways were human papillomavirus, lysosome, tryptophan metabolism, and tyrosine metabolism.

Table 2 Uniquely expressed whey proteins in donkey colostrum and mature Milk. UniProt accession

Description

peptide identification scores

Coverage (%)

Mol/Weight (kDa)

DC/DM

Q3ZCH5 A0A0A1E6K2 F7E0P3 Q8SPP7 F7DGX8 F7AAA8 E1BGJ0 F1MPP2 Q9MXD5 K9K2M0 H9GZN7 F1MW68 F7B847 G3MYD7 F1N2N5 G3X807 F6PZ29 F6VWX6 O97567 F6QIV8 F6XP08 F7D7M7 K9K4A2 H9GZZ3 Q9N2C4

Zinc-alpha-2-glycoprotein Immmunoglobulin lambda light chain variable region Thrombospondin 4 Peptidoglycan recognition protein1 Cartilageacidic protein 1 Peptidyl-prolylcis-transisomerase L receptorrelated protein 1 Insulin like growth factor binding protein7 MHC class II associated invariant chain DNA J-like protein subfamily B member 11-like protein Uncharacterized protein Cathepsin Z Amino peptidase Histone H3 Myristoylateda lanine-rich C-kinase substrate Histone H4 Multiple coagulation factor deficiency 2 C-Cmotifchemokine Transcription factor AEBP1 Follistatin like 1 ST6beta-galactoside alpha-2,6-sialyltransferase1 Uncharacterized protein Multiple coagulation factor deficiency protein-like protein Uncharacterized protein Glutathione peroxidase (Fragment)

133.78 143.85 46.25 145.25 130.29 54.09 23.47 101.57 155.08 34.75 103.34 57.09 77.30 110.90 97.77 48.98 144.17 53.04 92.87 133.32 88.24 100.92 63.98 63.62 106.42

56.2 21.9 3.1 50.5 36.5 25.7 0.5 49.3 6.2 13.4 47.8 41.4 3.9 35.3 4.5 53.1 68.3 14.2 12.5 3.9 6.8 2 68.5 11.6 37.2

33.851 23.398 102.89 21.063 70.935 19.597 504.76 29.05 23.425 27.793 7.3425 33.886 109.88 15.241 31.69 10.949 16.328 12.136 82.365 34.927 26.315 54.043 16.513 10.22 8.899

DC DC DC DC DC DC DC DC DC DC DC DC DC DM DM DM DM DM DM DM DM DM DM DM DM

4

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Fig. 1. Hierarchical clustering of whey proteins in donkey colostrum and mature milk (p < 0.05). The bar color represents a logarithmic scale from −2 to 2. Each column indicates a replicate experiment, and each row indicates a protein.

Fig. 2. GO annotation of differentially expressed whey proteins in donkey colostrum and mature milk. GO-BP, Gene ontology- biological process; GO-MF, Gene ontology- molecular function; GO-CC, Gene ontology- cellular component.

3.5. Protein–protein interaction network analysis of the differentially expressed whey proteins in donkey colostrum and mature milk

five interacting proteins, respectively. Other proteins had less than five interacting partners.

The protein-protein interaction network of whey proteins identified in donkey milk was analyzed by STRING software, version 10.5. The network contained 103 proteins. Among these, there were twelve differentially expressed or uniquely expressed proteins in donkey milk whey, and eight and four highly expressed proteins in colostrum and mature milk whey, respectively. As shown in Fig. 4, prothrombin (F7BFJ1) and apolipoprotein B(F6YCW8), interacting with eight proteins were the proteins that were found to interact the most, followed by L receptor-related protein 1 (E1BGJ0) and DNA J-like proteins Ub family B member 11-like protein fragment (K9K2M0), with seven and

3.6. Parallel reaction monitoring (PRM) analysis of the differentially expressed whey proteins in donkey colostrum and mature milk We established a parallel reaction monitoring method to verify the abundance of LC-MS/MS-scanned proteins by targeted proteomics. In PRM validation experiments, we selected to verify the abundance of five potential functional whey proteins in donkey milk. The results showed that PRM analysis could be successfully used to characterize the differentially expressed proteins in donkey colostrum and donkey constant milk. Compared with LC-MS/MS analysis, PRM analysis had 5

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Fig. 3. KEGG pathway analysis of differentially expressed whey proteins in donkey colostrum and mature milk.

quantitative comparisons between the colostrum and mature milk whey proteomes. In addition to its relevance to the understanding of the constituents of donkey milk, our study also advances the theoretical basis for the study of the milk proteomes of other species, especially non-ruminants, in the future. In a previous study, the major whey proteins in donkey milk were shown to include α-lactalbumin (α-LA), β-lactoglobulin (β-LG), and lysozyme (LYS), with the main components of whey protein being α-LA and β-LG, as in other species (Brumini, Criscione, Bordonaro, Vegarud, & Marletta, 2016; Cao et al., 2017). Lysozyme, as a key antimicrobial component in human milk, has the benefits of improving intestinal function and promoting body health (Brundige, Maga, Klasing, & Murray, 2008; Hennart, Brasseur, Delognedesnoeck, Dramaix, & Robyn, 1991; Maga et al., 2006). However, lysozyme is almost absent from dairy animal milk (Maga, Walker, Anderson, & Murray, 2006). In this study, we identified highly abundant lysozyme in donkey colostrum and mature milk whey. Previous studies have shown that donkey milk has a bacteriostatic effect on S. dysenteriae (Zhang, Zhao, Jiang, Dong, & Ren, 2008), and the active components of donkey milk could suppress tumor proliferation in vitro (Mao et al., 2009), which may be related to the high abundance of lysozyme in donkey milk whey. In our study, polymeric immunoglobulin receptor, lactotransferrin, and uncharacterized proteins (H9GZV1 and F6W1N4) were highly abundant in donkey whey. Polymerized immunoglobulin receptors play a key role in the immune system. They bind to polymerized IgA and IgM and transport them across cell membranes to perform immune functions (Kaetzel, 2010). The high abundance of polymerized immunoglobulin receptor in donkey milk whey protein can protect the human immune system, indicating its advantages over other animals’ milk as the base material for functional dairy products (Kaetzel, 2010; Li, Li, Zeng, Liu, & Ren, 2016). The other immunoglobulin proteins identified in our study included Igλ light chain variable region (A0A0A1E691), Ig λ light chain variable region (A0A0A1E417), Igγ 1 heavy chain constant region (Q95M34), Ig heavy constant mu (H9GZN9), and Igλ light chain variable region (A0A0A1E4I0). The above results show that whey protein of donkey milk has potential antibacterial and immune-promoting functions. In addition, other studies reported that highly abundant milk

higher sensitivity, accuracy, and reproducibility. As shown in Fig. 5 and Table 3, apolipoprotein B and ammonia oxidase, both upregulated proteins in colostrum whey, were confirmed as highly enriched in colostrum, while proteins (transforming growth factorβ and cathepsin B) that were upregulated in mature milk were also found to be highly enriched in mature milk. Immunoglobulin heavy constant mu was identified in both colostrum and mature milk, which was consistent with our identification results. 4. Discussion Whey protein, an important sub-component of the milk proteome, has many biological functions. Several mammalian whey proteomes have already been studied by advanced proteomic technology, and these studies provided a reference for our research (Cao et al., 2017; Elhatmi et al., 2015; Johnston, Estepa, Ebhardt, Crowe, & Diskin, 2018; Li et al., 2014, 2018; Liao, Alvarado, Phinney, & Bo, 2011; Yang et al., 2013, 2017). The whey proteomes of ruminants including buffalo, goat, cow, and camels, have been fully elucidated (Yang et al., 2013). A TMT proteomic approach has been used to explore 615 human and 580 buffalo whey proteins (Li et al., 2018), and iTRAQ applied to the proteins of cow, goat, buffalo, yak, and camel uncovered 211 whey proteins (Yang et al., 2013). Another study quantified 63 and 53 whey glycoproteins in human colostrum and mature milk respectively, using lectin enrichment and LC-MS/MS (Cao et al., 2017). These studies not only clarified the whey proteome of human and some mammalian milk, but also provided a theoretical basis for the study of other species’ whey proteomes (El-hatmi et al., 2015; Felfoul, Jardin, Gaucheron, Attia, & Ayadi, 2017; Medeiros et al., 2018). In addition, donkey whey proteomes have been identified in previous studies. Vincenzo Cunsolo et al. characterized and identified 106 gene products in donkey whey (Cunsolo et al., 2007, 2011). Therefore, the donkey milk proteome at various lactation stages has been not yet been comprehensively investigated. In the current study, we used a label-free proteomics approach combined with LC-MS/MS technology to quantify and identify the proteome of donkey milk whey. We identified 288 and 287 whey proteins in colostrum and normal milk respectively, and we made 6

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Fig. 4. Protein – protein interaction network analysis for differentially expressed Whey proteins between donkey colostrum and mature milk. Each node represents a protein, and each edge represents the interaction between proteins. Disconnected nodes are hidden.

biosynthesis of apolipoprotein B consumes microsomal triglyceride transporters to inhibit diet-induced obesity (Lee, Bao, Ishikawa, Wang, & Lim, 2017). Previous studies have also shown that donkey milk is suitable for people with dyslipidemia, and can promote weight loss (Martini et al., 2015; Ragona et al., 2016). In our study, the high relative amount of apolipoprotein B provides an explanation for the findings of previous studies. Not only because of the low fat content of donkey milk, but also because the high relative amount of apolipoprotein B in donkey colostrum whey protein function to regulate lipid metabolism. Cathepsin B, a lysosomal cysteine protease, is a member of the papain family and participates in physiological activities such as immune response (Mort & Buttle, 1997; Sloane, Dunn, & Honn, 1981). Changes in cathepsin B expression are often associated with cancer lesions. Previous studies have identified this protein has been in milk (Considine, Healy, Kelly, & McSweeney, 2004). In our study, cathepsin B was downregulated 3.7-fold in donkey colostrum whey compared with donkey mature milk. TGF-β is considered to be associated primarily with immunosuppressive functions and anti-inflammatory responses (Li, Wan, Sanjabi, Robertson, & Flavell, 2006; Mangan et al., 2006). It is well known that newborns, especially premature infants, are susceptible to intestinal inflammation and have a high mortality rate. TGF-β could reduce the production of proinflammatory factors, and

proteins identified in human and mammalian whey also included serum albumin, lactoferrin, xanthine dehydrogenase, and cluster proteins (Elhatmi et al., 2015; Li et al., 2018). In our study, these proteins were all identified in donkey colostrum and mature milk whey. Among the most abundant proteins, we also identified beta-casein, which may be due to residual casein from the whey protein separation process. Previous studies have found that the type, abundance, and modification of proteins varies between lactation stages (Cao et al., 2017; Yang et al., 2017). This may be due to the differences in the demand for protein types and contents for infants or young babies at different growth stages. Therefore, we compared the qualitative and quantitative changes of donkey whey protein with various different lactation stages using a label-free proteomic approach. In our study, we identified 18 differentially expressed whey proteins in donkey colostrum and donkey mature milk. Among these proteins, apolipoprotein B was found upregulated 17.15-fold (P = 0.0048) in donkey colostrum compared with mature milk. Apolipoprotein B, synthesized by the liver, and mainly located on the surface of low density lipoprotein (LDL), is a marker protein for atherosclerosis and other diseases, and plays a key role in cholesterol homeostasis (Walldius et al., 2002; Wang et al., 2018). A previous study showed that drosophila can regulate lipid metabolism through apolipoprotein B produced by cardiomyocytes, and that the 7

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F6YCW8

Peak urea

Peak urea

a 0.15 0.10

b

0.05

DC

DM

b

a

DM F1MZA0

30.00

0.60

a Peak urea

0.50

Peak urea

a

DC

F6YNH6

0.70

0.30

F6VB94

0.10 0.00

0.00

0.40

1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30

b

15.00

b

10.00 5.00

0.10

0.00

0.00 DC

DC

DM

DM

H9GZN9

a

Peak urea

10.00 8.00

b

6.00 4.00

0.00 DC

DM

Fig. 5. Parallel reaction monitoring analysis of selected proteins enriched in DC (F6YCW8, F1MZA0) or in DM (F6VB94, F6YNH6, H9GZN9). Average abundances were displayed with standard deviations.

we found that donkey milk whey contained high levels of TGF-β, and TGF-β was upregulated 2.85-fold in mature milk whey compared with colostrum milk. We also clarified the function of differentially expressed proteins in colostrum and mature milk. Eighteen differentially expressed and twenty-five uniquely expressed whey proteins in donkey colostrum and mature milk were categorized into three subgroups: biological processes, molecular functions, and cellular components. Previous studies have shown that human and some mammalian whey proteins are mainly involved in biological regulation, response to stimulus and immune system processes (Cao et al., 2017; Liao et al., 2011; Yang et al., 2017). However, the biological processes in which donkey whey proteins are involved have not yet been fully annotated. In our study, the differentially expressed proteins in donkey colostrum and mature milk whey not only participated in the above biological processes, but were also involved in metabolic processes and regulation of biological processes. It is worth noting that among the eleven whey proteins involved in immune system processing, nine were highly or uniquely expressed

Table 3 Comparison of LFQ and PRM quantification results. UniProt accession

Description

DC/DM (LFQ)

DC/DM (PRM)

F6YCW8 F6VB94

Apolipoprotein B Transforming growth factorβ induced Cathepsin B Amineoxidase Immunoglobulin heavy constant mu

17.15 0.35

4.15 0.68

0.27 3.76 1.00

0.58 2.80 0.85

F6YNH6 F1MZA0 H9GZN9

regulate intestinal epithelial permeability to inhibit the occurrence of enteritis by upregulating tight junction proteins, such as claudin-1 and claudin-4 (Rautava et al., 2011). Previous studies have identified that TGF-β in human whey, and human milk is considered an important source of TGF uptake in newborns (Rautava et al., 2011). In our study,

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5. Conclusions

in colostrum compared with mature milk. This result indicates that donkey colostrum whey proteins may be more active than mature milk in the establishment of the immune system. Previous studies have shown that calves fed colostrum within 24 h after birth not only gain a good immune status, but their metabolism is promoted (Hadorn, Hammon, Bruckmaier, & Blum, 1997). In our study, among the most common fifteen biological processes involved in whey differentially expressed proteins from donkey milk, eight were related to metabolic processes. Fifteen whey proteins in donkey colostrum were involved in the metabolic process, including five differentially expressed proteins and ten specifically expressed proteins. Amine oxidase (F1MZA0), Amine oxidase (F7CWC8), and Tetraspanin (F6Q9S8) were involved in all biological processes related to metabolism, and were upregulated 3.76-, 2.99- and 2.31-fold, respectively in donkey colostrum. Differentially expressed proteins in donkey colostrum whey may play an important role in the regulation of infant metabolism. According to the results of KEGG pathway analysis, differentially expressed proteins in donkey whey are mainly related to antigen processing and presentation, complement and coagulation cascades, the phagosome, and some diseases including malaria, systemic lupus erythematosus, and human papillomavirus infection. Major histocompatibility complexes (MHC) class I and class II molecules, and cathepsin B are involved in antigen processing and presentation. In a previous study (Elsen & Gobin, 1999; Ugolini & Vivier, 2001), MHC class I and class II molecules, such as glycoprotein, were identified in human colostrum and mature milk fat globule membrane. In our study, the MHC class I antigen (fragment) was upregulated 3.3-fold in colostrum milk whey and the MHC class II associated invariant chain was uniquely expressed in colostrum milk whey. Antigen receptor cells cannot directly recognize the antigen molecule, but react by recognizing the complex produced by the binding of antigen polypeptide and the MHC molecule. MHC class I and class II molecules may be related to the establishment of the neonatal immune system (Elsen & Gobin, 1999; Ugolini & Vivier, 2001). Previous studies reported that human, bovine, and buffalo milk whey proteins were mainly involved in two immune-related pathways: the complement and coagulation cascades, and the phagosome (Yang et al., 2013, 2017). In our study, differentially expressed proteins in donkey whey also participated in these two pathways. The phagosome is the vacuole in the plasma membrane, and contains invasive microorganisms engulfed by innate immune cells (Botelho, Scott, & Grinstein, 2004). As part of a pivotal pathway in innate and adaptive immunity, the phagosome plays a key role in eliminating apoptotic cells, host defense, inflammation, and tissue remodeling and repair (Gordon, 2016). The complement and coagulation cascades are interacting and co-activated systems (Amara et al., 2008; Oikonomopoulou, Ricklin, Ward, & Lambris, 2012; Yin et al., 2016). We identified thrombospondin 4, MHC class I antigen (fragment), prothrombin, and uncharacterized protein (F6XSF7) as differentially expressed whey proteins involved in the phagosome, complement, or coagulation pathways. These four proteins are highly expressed in the whey of donkey colostrum milk, further verifying the role of donkey colostrum whey proteins in immune function. We therefore obtained a preliminary understanding of the functions of donkey whey proteins with our KEGG pathway analysis. Protein interaction network analysis is of great significance to understanding the function of proteins and plays an important role in various biological processes. We found that, among the differentially expressed whey proteins in donkey milk whey, prothrombin and apolipoprotein B had the highest connectivity in the inferred protein interaction network. Compared with mature milk, prothrombin, involved in the complement and coagulation cascade pathway, was upregulated 4.14-fold in colostrum milk. It is worth noting that prothrombin was involved in ten abundant subgroups of biological processes, such as cellular processes, metabolic processes, stimulation reactions, and biological regulation.

In conclusion, we examined the donkey whey proteome and our work provides a basis for a better understanding of its composition, and the functions of its proteins. Of particular note, we found that donkey whey proteins are closely related to immune function and participate in a variety of disease-related metabolic pathways. This information will be helpful for the further study of the biological functions of donkey milk proteins. Our research results provide a certain basis for the comparative study of donkey milk and formula milk powder in the future. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by Shenyang Technological Innovation Project [grant number Y17-0- 028] and Liaoning Technological Innovation Team of Agricultural Science. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodres.2019.108741. References Amara, U., Rittirsch, D., Flierl, M., Bruckner, U., Klos, A., Gebhard, F., ... Huber-Lang, M. (2008). Interaction between the coagulation and complement system. Advances in Experimental Medicine and Biology, 632(2), 71–79. https://doi.org/10.1007/978-0387-78952-1_6. Botelho, R. J., Scott, C. C., & Grinstein, S. (2004). Phosphoinositide involvement in phagocytosis and phagosome maturation. Current Topics in Microbiology & Immunology, 282, 1–30. https://doi.org/10.1007/978-3-642-18805-3_1. Brendan, M. L., Tomazela, D. M., Shulman, N., Chambers, M., Finney, G. L., Frewen, B., ... Maccoss, M. J. (2010). Skyline: An open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics, 26(7), 966–968. https:// doi.org/10.1093/bioinformatics/btq054. Brumini, D., Criscione, A., Bordonaro, S., Vegarud, G. E., & Marletta, D. (2016). Whey proteins and their antimicrobial properties in donkey milk: A brief review. Dairy Science and Technology, 96(1), 1–14. https://doi.org/10.1007/s13594-015-0246-1. Brundige, D. R., Maga, E. A., Klasing, K. C., & Murray, J. D. (2008). Lysozyme transgenic goats' milk influences gastrointestinal morphology in young pigs. Journal of Nutrition, 138(5), 921–926. https://doi.org/10.1093/jn/138.5.921. Cao, X., Song, D., Yang, M., Yang, N., Ye, Q., Tao, D., ... Yue, X. (2017). Comparative analysis of whey N-glycoproteins in human colostrum and mature milk using quantitative glycoproteomics. Journal of Agricultural and Food Chemistry, 65, 10360–10367. https://doi.org/10.1021/acs.jafc.7b04381. Cao, X., Yang, M., Yang, N., Liang, X., Tao, D., Liu, B., ... Yue, X. (2019). Characterization and comparison of whey N-glycoproteomes from human and bovine colostrum and mature milks. Food Chemistry, 276, 266–273. https://doi.org/10.1016/j.foodchem. 2018.09.174. Codreanu, S. G., Hoeksema, M. D., Slebos, R. J. C., Zimmerman, L. J., Rahman, S. M. J., Li, M., ... Massion, P. P. (2017). Identification of Proteomic Features to Distinguish Benign Pulmonary Nodules from Lung Adenocarcinoma. Journal of Proteome Research, 16(9), 3266–3276. https://doi.org/10.1021/acs.jproteome.7b00245. Considine, T., Healy, Á., Kelly, A. L., & McSweeney, P. L. H. (2004). Hydrolysis of bovine caseins by cathepsin B, a cysteine proteinase indigenous to milk. International Dairy Journal, 14(2), 117–124. https://doi.org/10.1016/s0958-6946(03)00171-7. Contarini, G., Pelizzola, V., Scurati, S., & Povolo, M. (2017). Polar lipid of donkey milk fat: Phospholipid, ceramide and cholesterol composition. Journal of Food Composition and Analysis, 57, 16–23. https://doi.org/10.1016/j.jfca.2016.12.013. Cunsolo, V., Muccilli, V., Fasoli, E., Saletti, R., Righetti, P. G., & Foti, S. (2011). Poppea's bath liquor: The secret proteome of she-donkey's milk. Journal of Proteomics, 74(10), 2083–2099. https://doi.org/10.1016/j.jprot.2011.05.036. Cunsolo, V., Saletti, R., Muccilli, V., & Foti, S. (2007). Characterization of the protein profile of donkey's milk whey fraction. Journal of Mass Spectrometry, 42(9), 1162–1174. https://doi.org/10.1002/jms.1247. Cunsolo, V., Saletti, R., Muccilli, V., Gallina, S., Francesco, A. D., & Foti, S. (2017). Proteins and bioactive peptides from donkey milk: The molecular basis for its reduced allergenic properties. Food Research International, 99, 41–57. https://doi.org/10. 1016/j.foodres.2017.07.002.

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