2.2. Total Protein Extraction

After thawing the milk samples, 3 individual milk samples from each lactation stage were combined into 1 sample. This results in 4 biological replicates per lactation stage. Next, centrifuge the milk samples at 14,000× g and 4 °C for 15 min to remove milk lipids and cell impurities. Some samples were taken into an ultrafiltration tube (15 mL, 10 kD) and centrifuged at 14,000× g for 15 min. The ultra-filtered sample was put into a centrifuge tube and balanced with Dissolved Buffer (8 M Urea, 100 mM TEAB, pH 8.5) (Sigma, St. Louis, MI, USA). Then, take the supernatant and add 1 M DTT (Sigma, St. Louis, MI, USA) to react at 56 °C for 1 h, and subsequently alkylate with sufficient iodoacetamide in the dark at room temperature for 1 h. The protein quantity was tested with a Bradford protein quantitative kit.

2.3. Trypsin Treatment

Dissolved Buffer was added to the protein sample to 100 μL. After that, trypsin (Promega, Madison, WI, USA) and a 100 mM TEAB buffer (Sigma, St. Louis, MI, USA) were added, and the sample was mixed and digested at 37 °C for 4 h. Next, add the trypsin and CaCl₂ to digest overnight. Mix formic acid with the digested sample, adjust the pH to under 3, and centrifuge at 12,000× g for 5 min. Slowly load the supernatant onto a C18 desalination column, wash it three times with a washing buffer (0.1% formic acid, 3% acetonitrile), and then add an elution buffer (0.1% formic acid, 70% acetonitrile). Collect the eluents of each sample for lyophilization.

2.4. DDA Spectrum Library Construction

DDA spectrum library construction and DIA mode identification using UHPLC-MS/MS were performed.

2.4.1. Separation of Fractions

Mobile phases A (2% acetonitrile, pH 10.0) and B (98% acetonitrile, pH 10.0) were used for gradient elution (Table 1 and Table 2). Dissolve the lyophilized powder (200 ng) in solution A and centrifuge at room temperature at 12,000× g for 10 min. Use C18 columns (Waters BEH C18, 4.6 × 250 mm, 5 μm) to fractionate the sample on the Rigol L3000 HPLC system with a column oven set at 45 °C. Monitor the eluates at UV 214 nm, collect 1 tube per minute, and finally combin into 4 or 6 fractions. The chromatogram of the fractions collected was shown in Figure 1. Dry all fractions under a vacuum and reconstruct them in 0.1% (v/v) formic acid (FA) in water.

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Figure 1

The chromatogram of the fractions collected.

Table 1

Four of peptide fractions separation liquid chromatography elution gradient table.

Time (min)	Flow Rate (mL/min)	Mobile Phase A (%)	Mobile Phase B (%)
0	1	97	3
10	1	95	5
20	1	80	20
27	1	60	40
29	1	50	50
30	1	30	70
35	1	0	100

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Table 2

Six of peptide fractions separation liquid chromatography elution gradient table.

Time (min)	Flow Rate (mL/min)	Mobile Phase A (%)	Mobile Phase B (%)
0	1	97	3
10	1	95	5
12	1	90	10
13	1	85	15
50	1	60	40
53	1	40	60
54	1	0	100
58	1	0	0
60	1	0	0
70	1	0	0

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3.2. Identification of DEPs in Different Lactation Stages

The number of identified DEPs in DM in different lactation stages is shown in Table 3. The volcano plots of DEPs in milk from 1, 30, 60, 90, 150, and 180 d of dairy donkeys are shown in Figure 3. The DEPs in DM between different lactation stages are shown in Table S2. The subcellular localization of the DEPs is shown in Figure 4. Taking the B vs. A comparison group as an example, a total of 327 candidate DEPs, including 161 up- and 166 down-regulated DEPs, were obtained from the GO and metabolic pathway analysis.

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Figure 3

Volcano plots of DEPs in DM in different lactation stages using a data-independent acquisition (DIA)-based proteomics approach. FC = fold change. Note: milk samples were collected from Dezhou donkeys at seven different lactation stages: 1 d (A), 30 d (B), 60 d (C), 90 d (D), 120 d (E), 150 d (F_), and 180 d (G) after foaling.

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Figure 4

Subcellular localization of DEPs in the milk in different lactation stages. Note: milk samples were collected from Dezhou donkeys at seven different lactation stages: 1 d (A), 30 d (B), 60 d (C), 90 d (D), 120 d (E), 150 d (F_), and 180 d (G) after foaling.

The relative expression of DM proteins (whey protein and casein) in different lactation stages was analyzed. The results showed that whey protein in DM mainly included α-lactalbumin, β-lactoglobulin, albumin, lysozyme, and lactoferrin, and casein mainly included κ-casein, β-casein, α-s1-casein, and α-s2-casein. The expression level of all the whey proteins and casein in mature milk (groups B, C, D, E, F_, and G) was higher than colostrum (group A) (Figure 5). With the extension of the lactation period, the relative expression level of lactoferrin increased. The relative expression levels of other whey proteins (albumin, lysozyme, β-lactoglobulin, and α-lactalbumin) gradually increased within 60 days of lactation and then reached the plateau stage (Figure 5A). As shown in Figure 5B, the level of α-s1-casein increased in the early lactation stage, decreased in the middle to late lactation stages, and then increased again on the 180th day of lactation (Figure 5B). The relative expression level of α-s2-casein and κ-casein increased and then decreased with the extension of the lactation period. β-casein gradually increased within 30 days of lactation and then remained stable (Figure 5B).

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Figure 5

The expression levels of whey protein (A) and casein (B) in DM in different lactation stages related to colostrum. Note: milk samples were collected from Dezhou donkeys at seven different lactation stages: 1 d (A), 30 d (B), 60 d (C), 90 d (D), 120 d (E), 150 d (F_), and 180 d (G) after foaling.

3.3. GO Analysis of DEPs

The DEPs between the different lactation stages in DM were classified into CC, MF, and BP, according to GO annotation terms. Take B. vs. A, for example. GO analysis revealed that the 327 DEPs were mainly related to BP, such as blood coagulation, hyaluronan metabolic process, and the single multicellular organism process; and MF, such as endopeptidase activity and serine-type endopeptidase activity (Table 4).

4.1. Nutrition and Transportation

There was more selenoprotein F (FC > 6) in mature milk in the mid and late lactation (90–180 d) stage. Selenium (Se) is one important essential trace element in humans and animals. It is a structural component with many proteins and plays a crucial role in physiological processes such as DNA synthesis, the scavenging of toxins, antioxidant defense, and thyroid hormone metabolism [26,27]. Se deficiency can cause reproductive disorders (such as abortion and infertility) and muscular diseases (such as white muscle disease) [27]. However, excessive Se can cause animal poisoning [28]. Compared to inorganic Se, organic Se is considered a safe and effective bioavailable source for humans and animals [27,29]. Zhang et al. (2020) studied the hypotensive and immune-enhancing components in buffalo whey at different altitudes [30]. It was found that the selenoprotein F content of whey at low altitudes was higher than whey at high altitudes, and buffalo milk at low altitudes was suitable for producing immune-regulation milk [30]. DM also had a high content of Se, which was beneficial for normal immune system function [31,32]. The result of this study showed that DM from mid and late lactation had a high content of selenoprotein F. Therefore, DM from mid and late lactation can serve as a good source of organic Se supplementation.

In addition, the contents of some transporters, such as transcobalamin 1 (TC, FC > 26) and solute carrier family 36 member 1 (SLC36A1, FC > 19), were also high in mature milk (30–180 d). Milk is an abundant source of bioavailable vitamin B₁₂ (VB₁₂). However, the bioavailability of VB₁₂ in milk is influenced by many factors, such as transport carriers and intestinal uptake capacity. TC is a VB₁₂-specific binding protein, which may influence VB₁₂ bioavailability. Fedosov et al. (2019) investigated the effects of vitamin carriers on VB₁₂ nutritional availability in cow and buffalo milk and found that VB₁₂ bioavailability was higher in CM (with TC-VB₁₂) than buffalo milk (with haptocorrin-VB₁₂) [33]. The research by Hine et al. (2014) found that bovine milk TC can stimulate bovine, mouse, and human intestine VB₁₂ uptake [34]. The TC-VB₁₂ complex extracted from milk can be used as a natural biological source for VB₁₂ to overcome the malabsorption of VB₁₂ [34]. Our study found that donkey mature milk (30 d–180 d) contained abundant TC. In terms of the bioavailability of VB₁₂, DM and CM were more beneficial for human health [35]. SLC36A1 is a neutral amino acid transporter. It exerts a variety of important physiological functions, including promoting nutrient absorption, controlling nutrient recycling, and activating the mTORC1 signaling pathway [36]. In a study by Wang et al. (2021), SLC36A1 mediated the activation of mTORC1 induced by leucine [37]. Therefore, SLC36A1 in DM may play a role in the nutrient’s absorption and signal transduction, which requires further research.

4.2. Cell Proliferation and Differentiation

There were also some cell proliferation and differentiation associated components, such as transforming growth factor-beta-induced protein ig-h3 (beta ig-h3, FC > 24), sushi repeat-containing protein X-linked (SRPX, FC > 22), secreted phosphoprotein 1 (SPP1, FC > 7), tyrosine–protein kinase (FC > 11), and milk fat globule-EGF factor 8 protein (Mfge8, FC > 5) in mature milk (30–180 d). Among them, beta ig-h3, SRPX, and SPP1 were extracellular matrix-associated proteins, while tyrosine–protein kinase and Mfge8 played a role in cell proliferation, differentiation, and apoptosis.

Beta ig-h3 is an extracellular matrix protein that exists in many tissues, such as the kidneys, spleen, bladder, and lungs, and can be detected in culture media of human lung and bladder smooth muscle cells and fibroblasts cultured in vitro [38]. Beta ig-h3 is involved in the cell’s adhesion, migration, proliferation, differentiation, and apoptosis [39,40]. SRPX is a chondroitin sulfate proteoglyean that belongs to extracellular matrix protein. It can promote synaptogenesis in the cerebral cortex, regulate the development of neurons, and regulate the immune system [41]. SPP1 is a multifunctional protein that plays a role in intercellular communication and extracellular matrix, with functions of cytokines, chemokines, and signal transduction [42]. It plays an important role in disease (cardiovascular disease, cancer, diabetes, and kidney stone disease), inflammation, and wound healing processes [43]. The presence of these extracellular matrix-related proteins in DM was beneficial for the proliferation and differentiation of intestinal cells in donkey foals and infants.

Tyrosine–protein kinases can phosphorylate the protein tyrosine residues and play a very important role in cell growth and differentiation. The bioactive substances in HM had protective effects on infants. HM can induce the proliferation of fetal small intestine cells, which depend on the signaling pathway involved in tyrosine–protein kinase [44]. In addition, tyrosine–protein kinase also collaborates with Mfge8 to participate in the clearance of apoptotic cells. Mfge8 can eliminate apoptotic cells by regulating phagocytes and plays an important role in self-stabilization, angiogenesis, inflammation, and other processes. It was reported that tyrosine kinase and Mfge8 were up-regulated during inflammation and synergistically cleared damaged cells, which was beneficial for repairing dysfunctional tissues [45]. Thus, a high expression of tyrosine–protein kinase and Mfge8 in DM may promote the growth and proliferation of small intestine cells and repair damaged intestinal tissue, which is beneficial for the intestinal function of donkey foals and infants.

4.3. Antibacterial and Anti-Inflammatory Activity

There were some enzymes such as alpha-mannosidase (FC > 18), alpha-1,2-Mannosidase (FC > 8), Peroxiredoxin 4 (Prdx4, FC > 17), cathepsin B (FC > 10), and lysozyme (FC > 9) in donkey mature milk (30–180 d), which played important roles in antibacterial and anti-inflammatory activity. Alpha-mannosidases are widely expressed in human and animal tissues and body fluids [46]. They play an important role in glycoproteins metabolism, such as protein glycosylation and hydrolysis of glycoproteins. Alpha-mannosidases were mainly involved in the folding, maturation, transportation, and biological activity of newly formed glycoproteins, and thus play a role in cell adhesion, inflammatory response, and immune monitoring [47]. It was reported that alpha-mannosidase digested the biofilm and allowed neutrophils to kill the bacteria in infected mice [48]. Prdx4 is a vital antioxidant in cells that can hydrolyze hydrogen peroxide and promote oxidative protein folding [49]. Prdx4 plays a critical role in inflammatory diseases. It was reported that Prdx4 prevented inflammation and cell apoptosis by inhibiting oxidative stress [50]. A study by Yamada et al. (2012) found that the overexpression of human Prdx4 in transgenic mice can protect the pancreatic beta-cells from damage (insulitis) by inhibiting oxidative stress and inflammatory signals [51]. Cathepsin B is a lysosomal cysteine protease. It plays a role in protein catabolism and may participate in some physiological processes such as antigen processing and presentation, hormone activation, apoptosis, aging, autophagy, and bone turnover [52,53]. In addition, a lysozyme is a kind of hydrolase that can hydrolyze peptidoglycan, the main component of bacterial cell walls. Proteomic analysis of DM showed that lysozyme had antibacterial activity [6]. It was found that the oral administration of heat-treated DM with preserved lysozyme activity can improve intestinal injury induced by chronic stress in mice [54]. The results of other studies show that a lysozyme, together with lactoferrin and immunoglobulin, can inhibit the growth of gastrointestinal pathogenic microorganisms and reduce the incidence of gastrointestinal infection in infants [12]. These results indicated that DM had potential biological activities, such as antioxidant activity, immune stimulation, anti-inflammatory, and antibacterial properties (Figure 2 and Figure 6). DM is an ideal source of nutrition for growing children, convalescent patients, and the elderly [4,6].