Mohn Li, Qilong Li, Hikun Yu, Xiumin Zhng, Deho Li,Wnying Song, Yn Zheng,*, Xiqing Yue,*

a College of Food Science, Shenyang Agricultural University, Shenyang 110866, China

b College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China

c Beijing Academy of Food Sciences, Beijing 100068, China

d The Fourth Aff iliated Hospital of China Medical University, Shenyang 110866, China

Keywords:Bovine colostrum Donkey colostrum Proteomics Whey protein Gene Ontology Kyoto Encyclopedia of Genes and Genomes

A B S T R A C T This study aimed to analyze and compare the differentially expressed whey proteins (DEWPs) of donkey and bovine colostrum using high-performance liquid chromatography with tandem mass spectrometry-based proteomics. A total of 620 and 696 whey proteins were characterized in the donkey and bovine colostrum,respectively, including 383 common whey proteins. Among these common proteins, 80 were identified as DEWPs, including 21 upregulated and 59 downregulated DEWPs in donkey colostrum compared to bovine colostrum. Gene Ontology analysis revealed that these DEWPs were mainly related to cellular components,such as extracellular exosome, plasma membrane, and mitochondrion; biological processes, such as oxidation-reduction process, cell-cell adhesion, and small guanosine triphosphate (GTP) ase-mediated signal transduction; and molecular functions, such as GTP binding, GTPase activity, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor activity. Metabolic pathway analysis suggested that the majority of the DEWPs were associated with soluble NSF factor attachment protein receptor interactions in vesicular transport,fatty acid biosynthesis, and estrogen signaling pathways. Our results provide a vital insight into the differences between donkey and bovine colostrum, along with important information on the significant components as nutritional and functional factors to be included in infant formula based on multiple milk sources.

1. Introduction

Colostrum is the first form of milk produced by the mammary glands of mammals immediately following delivery of a newborn [1,2].Colostrum contains antibodies, which can protect the newborn against disease and infection, as well as immune and growth factors and other bioactive compounds, which help to activate the newborn’s immune system, jumpstart gut function, and seed a healthy gut microbiome in the first few days of life [3,4]. Bovine and human colostrum have a very similar composition, including many of the same antibodies, immune and growth factors, and other nutrients [5,6]. Bovine colostrum shows several benef its for human health, including supporting immune health [7], digestive health [8],early life nutrition [9], and sports nutrition [10]. However, bovine milk is also one of the most common food allergens responsible for allergies in infants [11,12]. Approximately 5%-15% of infants experience an allergic reaction from a bovine milk protein allergy,and this incidence is likely underestimated given the difficulty in testing for bovine milk allergy [13]. Donkey milk is suggested to be

an ideal substitute because of its similar protein and lactose content to human milk [14,15]. Moreover, donkey milk contains more whey proteins (approximately 35%-50%) than bovine milk (approximately 20%) [16], indicating its suitability as a potential substitute for bovine milk in infants with an allergy to bovine milk protein [17,18].

Whey protein is a major milk protein that plays a vital role in the immune defense of newborn mammals via homologous transfer [19,20]. Whey proteins mainly includeα-lactalbumin,β-lactoglobulin, serum albumin, lactoperoxidase, immunoglobulins,and lactoferrin [21]. Donkey milk is characterized by low levels of casein, with a whey/casein protein ratio almost equivalent to that of human milk compared to that of bovine milk [17]. These characteristics are thought to determine the remarkable differences in allergenicity and digestibility between donkey and bovine milk [16,22].Additionally, studies have shown that donkey whey proteins have certain physiological functions, such as antimicrobial [23] and anticancer properties [24-26].

Compared to the characterization of bovine milk protein, the research on donkey milk protein, including whey, casein, and milk fat globule membrane (MFGM) protein, is relatively lacking.Chianese et al. [27] investigated donkey casein using a proteomics approach, which involved one- and two-dimensional electrophoresis,staining with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural mass spectrometry (MS) analysis, and ultimately observed eleven components ofκ-casein, 6 phosphorylated components ofβ- andαS1-casein, and 3 main phosphorylated components ofαS2-casein. Zhang et al. [28] characterized donkey whey proteins from different milk yields of Dezhou donkeys using a label-free based comparative proteomics approach and identified 216 whey proteins, 19 of which showed significant differences in high-milk-yield samples. Li et al. [29] identified and quantified 288 and 287 donkey whey proteins in donkey colostrum and mature milk, respectively. Furthermore, Li et al. [30]also characterized 947 MFGM proteins, including 902 and 913 MFGM proteins in donkey colostrum and mature milk,respectively. It can be seen that most studies only focus on the comparison of donkey milk protein among species, whereas studies on the comparison of milk protein among different species, especially the comparison of donkey colostrum and bovine colostrum whey protein, remain very limited.

Thus, the purpose of this study was to characterize and compare whey proteins from donkey and bovine colostrum using a high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS)-based proteomics approach. We further aimed to identify the differentially expressed whey proteins (DEWPs)among proteins common to both donkey and bovine colostrum as well as investigate their interactions and related metabolic pathways.The results may contribute to gaining a deeper knowledge of the composition of whey proteins and provide a theoretical basis for the optimization of colostrum products, especially for the development of infant formula by mixing the two aforementioned milk sources.

2. Materials and methods

2.1 Sample collection

Donkey and bovine colostrum were collected from 45 Dezhou donkeys and 45 Holstein cows, respectively, at a local farm in Shenyang, China. All colostrum samples were collected from healthy donkeys or cows aged 2-3 years in the first 24 h after delivery.The 45 samples from each source were divided into 3 groups (with 15 colostrum samples each), and the samples from each group were pooled before the experiment. The experiment was repeated 3 times to exclude individual differences. Samples were stored at-80 °C until analysis.

2.2 Preparation of whey fractions

Colostrum samples were centrifuged (15 min, 10 000 ×g,4 °C) to remove milk lipids and cell impurities. Formic acid (33%)was then added to each centrifuged sample, and the pH of the centrifuged sample was adjusted to 4.6 with 3.3 mol/L sodium acetate(30 μL). The sample was then centrifuged (20 min, 14 000 ×g,4 °C) to remove casein from the mixture. The supernatant obtained following centrifugation contained the whey fraction separated from the colostrum sample. The concentration of whey protein was measured using bicinchoninic acid protein assays (P0012;Beyotime, Beijing, China).

2.3 Trypsin digestion of whey fractions

Whey proteins (600 μg) were added to a solution comprising 4% sodium dodecyl sulfate, 100 mmol/L Tris-HCl (pH 8.0), and 100 mmol/L dithiothreitol, followed by heating at 90 °C for 10 min. The samples were statically placed and cooled to 20 °C,and then loaded onto a 10 kDa ultrafilter (Millipore, Bedford, MA,USA) containing 200 μL UT buffer (8 mol/L urea and 150 mmol/L Tris-HCl; pH 8.0). The sample mixture was centrifuged (30 min,14 000 ×g, 4 °C) and washed with UT buffer. Iodoacetamide(final concentration of 50 mmol/L in UT buffer) was added to the ultrafilter and mixed with the sample for 1 min. The sample mixture was transferred to a dark environment, incubated at 20 °C for 30 min, centrifuged at 14 000 ×gfor 15 min at 4 °C, and washed with 100 μL UT buffer. This step was repeated twice. The filter was then pre-washed by adding the dissolution buffer (UT buffer and 25 mmol/L NH4HCO3). The protein was digested at 37 °C for 24 h by adding 40 μL of trypsin to the filter. Digested samples were centrifuged (15 min, 14 000 ×g, 4 °C), and the peptide segments were collected for analysis. The absorbance of the peptides was then measured at 280 nm using an ultraviolet spectrophotometer.

2.4 HPLC-MS/MS

Sample peptide fragments were separated using a Thermo Fisher Scientific EASY-nLC 1000 HPLC system (Waltham, MA, USA),and then the proteins were identified by MS on a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). The separation system consisted of mobile phase A (0.1% formic acid and 99.9% water)and mobile phase B (84% acetonitrile, 0.1% formic acid, and 15.9%water). The chromatography column was equilibrated for 30 min using 95% mobile phase A. The mixture of peptide segments was loaded onto the upper column (Thermo Fisher Scientific Acclaim PepMap100, 100 μm × 2 cm, 3 μm, nanoViper C18) using an automatic sampler. Subsequently, the samples were separated using an analytical column (Thermo Fisher Scientific EASY column, 10 cm × 75 μm,3 μm, C18-A2) at 300 nL/min. Mobile phase B was used to separate the mixture of peptide segments. The separation time was set to 2 h under the following conditions: 0%-55% B from 0 min to 110 min, increase to 100% B at 115 min, and retain the liquid-phase gradient of B at 100% until 120 min. MS analysis was performed in positive ion detection mode for 120 min, with a scanning range of parent ions set tom/z300-1 800. The first-order MS resolution of the Q-Exactive system was set to 70 000 atm/z200, and 1 × 106was set as the automatic gain control target. The maximum isolation time was 50 ms, and the dynamic exclusion operation was conducted at 60 s. MS/MS data were obtained from the first 20 fragment maps with charge ≥ 2 collected from each full scan. The MS2activation type was set to higher-energy collisional dissociation,and an isolation window with a mass-to-charge ratio ofm/z2 was selected. Second-order MS was conducted at a spectrum ofm/z17 500 and 200. The normalized collision energy was 30 eV, and the underfill ratio was 0.1%.

2.5 Analysis of MS raw data and identification of proteins

MaxQuant software (version 1.5.3.17) was used to analyze the raw MS data and identify the proteins. Trypsin was used as the lyase,and two maximum missed cleavages were allowed for the database search. Peptide mass tolerance and MS/MS tolerance were set at 20 ppm and 0.1 Da, respectively. Formamide methyl was used for fixed modifications, and oxidation was used for variable modifications. Reversed versions of the target database were used as the database pattern to calculate the false discovery rate (FDR).The screening criteria for proteins and peptides were those identified with ＞ 99% confidence at an FDR ≤ 1%.

2.6 Bioinformatics and statistical analyses

Gene Ontology (GO; http://geneontology.org/) annotation [31],Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) pathway analysis [32], and the Database for Annotation, Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov/home.jsp) tool [33] were used to analyze the related metabolic pathways and functional categories of whey proteins in donkey and bovine colostrum. DEWPs between donkey and bovine colostrum were identified according to a thresholdP＜ 0.05 and fold change ≥ ± 1.50 in terms of protein expression levels. To investigate whether the metabolism of DEWPs differed between donkey and bovine colostrum, the protein counts andPvalues were calculated from the pathway topology analysis. The DEWPs were further analyzed using STRING (https://string-db.org)to obtain a protein-protein interaction network map.

3. Results

3.1 Characterization of whey proteins in donkey and bovine colostrum

In total, 933 whey proteins were characterized (Table S1),including 620 and 696 whey proteins in donkey and bovine colostrum,respectively. Among them, 383 common whey proteins (41.1%) were found in both groups. A total of 237 donkey-specific whey proteins(25.4%) and 313 bovine-specific whey proteins (33.5%) were also found (Fig. 1A).

Fig. 1 (A) Venn diagram of the identified whey proteins in donkey and bovine colostrum. (B) Volcano plot of the identified whey proteins in donkey and bovine colostrum. (C) Heat map analysis of the differentially expressed whey proteins between donkey and bovine colostrum. DC, donkey colostrum; BC, bovine colostrum.

3.2 Identification of DEWPs between donkey and bovine colostrum

The identified differences in whey proteins between donkey and bovine colostrum laid the foundation for DEWP screening. The subsequent analysis focused on the 383 common whey proteins that are shared by donkey and bovine colostrum and attempted to screen out DEWPs from these common whey proteins. Thus, a threshold was set to screen for DEWPs between donkey and bovine colostrum. In this study, aP＜ 0.05 and fold change ≥ ± 1.50 were used as thresholds. As shown in Fig. 1 and Table 1, 80 DEWPs were identified among the 383 common whey proteins between donkey and bovine colostrum, including 21 upregulated and 59 downregulated DEWPs in donkey colostrum.

Table 1 (Continued)

Table 1Differentially expressed whey proteins in donkey and bovine colostrum.

3.3 GO analysis of DEWPs

The 80 DEWPs between donkey and bovine colostrum were classified into 3 subcomponents (Fig. 2) according to GO annotation terms: cellular components (CC), molecular functions (MF), and biological processes (BP). The CC subgroup, which contained the most DEWPs, included extracellular exosome, plasma membrane,mitochondrion, cytosol, membrane, endoplasmic reticulum, solubleN-ethylmaleimide-sensitive factor (NSF) attachment protein receptor(SNARE) complex, lipid particles, focal adhesion, and endoplasmic reticulum membrane. The MF subgroup contained guanosine triphosphate (GTP) binding, GTPase activity, soluble NSF attachment protein (SNAP) receptor activity, cadherin binding involved in cell-cell adhesion, protein binding, SNARE binding, calciumdependent phospholipid binding, guanosine diphosphate binding,signal transducer activity, and phospholipase A2 inhibitor activity.Furthermore, the most common BP terms were oxidation-reduction process, cell-cell adhesion, small GTPase-mediated signal transduction, cholesterol biosynthetic process, fatty acid biosynthetic process, positive regulation of vesicle fusion, dopamine receptor signaling pathway, positive regulation of sequestering of triglycerides,synaptic vesicle fusion to presynaptic active zone membrane, and mammary gland development.

Fig. 2 GO annotation of the differentially expressed whey proteins between donkey and bovine colostrum.

Fig. 3 Metabolomic pathways view map of the differentially expressed whey proteins between donkey and bovine colostrum.

3.4 Metabolic pathway analysis of DEWPs

The top 20 major metabolic pathways related to the DEWPs are shown in Fig. 3. SNARE interactions in vesicular transport were the most relevant pathways, followed by fatty acid biosynthesis, estrogen signaling pathway, toxoplasmosis and chagas disease (American trypanosomiasis), platelet activation, fatty acid metabolism,legionellosis, long-term depression, biosynthesis of antibiotics,glucagon signaling pathway, Rap1 signaling pathway, circadian entrainment, melanogenesis, retrograde endocannabinoid signaling,cholinergic synapse, steroid biosynthesis, glutamatergic synapse,serotonergic synapse, and AMP-activated protein kinase signaling

pathways.

3.5 Protein-protein interaction network of DEWPs

As shown in Fig. 4, the protein-protein interaction network included 63 proteins and 108 interactions. Annexin A2 (P04272),vesicle-associated membrane protein 8 (VAMP8; Q3T0Y8), and lipopolysaccharide receptor (Q5XWB8), which interact with 8 proteins, were found to represent a node with the most interactions,followed by alpha-soluble NSF attachment protein (A5D7S0) and synaptobrevin homolog YKT6 (Q3T000) with 7 interactions.

Fig. 4 Protein-protein interaction network analysis of the differentially expressed whey proteins between donkey and bovine colostrum.

4. Discussion

In this study, 237 and 313 unique whey proteins in donkey and bovine colostrum, respectively, were found. This difference may be due to the significantly different signal pathways of protein metabolism and milk protein secretion in ruminants and monogastric animals. A large number of microorganisms in the rumen of ruminants can degrade and synthesize proteins as well as have the ability to better utilize dietary proteins, non-protein nitrogen, and regenerated urea as nitrogen sources [34,35]. Other studies have also found significant differences between donkey and bovine milk lipids [36-38],mainly due to the different fatty acid precursors in ruminants [39,40].These characteristics of ruminants can also explain why donkey milk shares more similarities with human milk compared to bovine milk to a certain extent. Among the 383 shared whey proteins in both donkey and bovine colostrum, we found 80 DEWPs (P＜ 0.05 and fold change ≥ ± 1.50), including 21 upregulated and 59 downregulated DEWPs in donkey colostrum. Among them,β-lactoglobulin-2 (P19647)was the protein with the highest fold change value (upregulated),followed byβ-lactoglobulin-1 (P13613).β-Lactoglobulin is a milk-specific protein synthesized by mammary epithelial cells and the main whey protein component in the milk of ruminants and pigs,horses, donkeys, and other animals, whereas its content in human milk is very low [41,42].β-Lactoglobulin is a lipocalin protein that can bind many hydrophobic molecules, suggesting a role in their transport [43]. In addition,β-lactoglobulin binds iron via siderophores and thus might play a role in combating pathogens [44]. Interestingly,many studies have shown that equine animals, including donkeys,have strong innate and adaptive immunity [45]; however, whether this is related to the higher content ofβ-lactoglobulin ingested from colostrum remains to be explored.

The functions and biological processes that the DEWPs may participate in were also clarified using GO annotation. Most of the DEWPs were found to participate in the CC subgroup, particularly the extracellular exosome, followed by the plasma membrane and mitochondrion. Cells naturally release extracellular exosomes and vesicles, which carry cellular proteins, nucleic acids, lipids,metabolites, and organelles as cargo [46,47]. Thus, the identified DEWPs are likely to be secreted by the heterogeneous cell populations of milk, including milk secretory cells, myoepithelial cells, and a hierarchy of progenitor and stem cells [48], which then participate in formation of extracellular exosomes and vesicles that enter the milk.However, further research is needed confirm this hypothesis and uncover the underlying mechanism.

Pathways involving SNARE interactions in vesicular transport were among the most relevant pathways associated with the DEWPs,followed by fatty acid biosynthesis and the estrogen signaling pathway.SNARE constitutes a large class of proteins present in all organelles involved in the transport and secretion of intracellular vesicles and can participate in endocytosis and phagocytosis [49]. Dietary bovine milk exosomes and their cargo are transported to the peripheral tissues via human vascular endothelial cells by endocytosis, which depends on cell and exosome surface glycoproteins in human intestinal cells [50,51].Our results demonstrate that the extent to which proteins and other nutrients are involved in endocytosis vastly differs between colostrum types, which provides new research directions for gene regulation targeting dietary nucleic acids across species boundaries from different colostrum sources. However, we have not verified the specific expression levels of differential and key proteins in various metabolic pathways, which is the direction of our future research.

Furthermore, the protein-protein interaction network showed that the DEWPs P04272, Q3T0Y8, and Q5XWB8 each interacted with eight proteins and represented a node with the most interactions.Q3TOY8, which is also known as VAMP8 or endobrevin, participates in endocytosis and regulates exocytosis in pancreatic acinar cells [52].VAMP8 interacts specifically with SNAP, most likely through a VAMP8-containing SNARE complex [53], which further supports the accuracy and consistency of our GO and metabolic pathway analyses.Overall, the results of the metabolic pathway and protein-protein interaction network analyses of DEWPs provides new insight into the functions of proteins between different species, which further reflect the specific needs of different mammalian newborns. These results may provide significant information on the composition of colostrum whey, which can offer insight into the nutritional and functional requirements of these important ingredients for the development of infant formula based on multiple milk sources.

5. Conclusions

Here, the whey proteins of donkey and bovine colostrum were investigated using label-free quantitative proteomics. A total of 933 whey proteins were identified, including 383 common proteins in both groups, 237 specific proteins in donkey colostrum, and 313 specific proteins in bovine colostrum. A total of 80 DEWPs were selected among the 383 common proteins, and the GO annotations, metabolic pathways, and interaction networks of these DEWPs were further determined. These results may provide significant information on the composition of colostrum whey as well as vital information on the nutritional and functional requirements of these important ingredients for infant formula development based on multiple milk sources.

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgments

This work was supported by the by National Key R & D Program of China (2018YFC1604302), “Twelfth Five Year” National Science and Technology Plan Project (2013BAD18B03), Chinese Scholarship Council (202008210391), Shenyang Technological Innovation Project (Y17-0-028), and LiaoNing Revitalization Talents Project(XLYC1902083). We thanked Dalian “Shenghongdaoda” and Tieling“Xingfazhongchu” farm for providing milk samples.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://doi.org/10.1016/j.fshw.2022.10.004.