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Comparative analysis of changes in whey proteins of goat milk throughout the lactation cycle using quantitative proteomics

Open AccessPublished:November 21, 2022DOI:https://doi.org/10.3168/jds.2022-21800

      ABSTRACT

      The composition and content of goat milk proteins are affected by many factors and have been extensively studied. However, variation in whey protein composition in goat milk throughout the lactation cycle has not been clarified. In the current study, 15 dairy goats were selected, and milk samples were collected at 1, 3, 30, 90, 150, and 240 d after delivery. Whey proteins were separated and digested and then identified using data-independent acquisition (DIA) and data-dependent acquisition proteomics approaches. Protein profiles identified using DIA were consistent with those of the data-dependent acquisition proteomics approach according to clustering and principal component analyses. Significant differences in the abundance of 238 proteins around the lactation cycle were identified using the DIA approach. Developmental changes of the whey proteome corresponding to lactation stage were revealed: plasminogen, α-2-macroglobulin, and fibronectin levels decreased from d 1 to 240, whereas polymeric immunoglobulin receptor, nucleobindin 2, fatty acid-binding protein 3, and lactoperoxidase increased from d 1 to 240. Protein-protein interaction analysis showed that fibronectin with a higher degree of connectivity is a central node. The findings are of great significance to better understanding the potential role of specific proteins and the mechanism of protein biosynthesis or intercellular transport in the mammary glands related to the physiological changes of dairy goats.

      Key words

      INTRODUCTION

      Milk is rich in nutrients, including proteins, fats, vitamins, and minerals. Although the essential components of protein, fat, and TS are similar in both goat and bovine milk, goat milk has lower αS1-casein content and is considered less allergenic than bovine milk (
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      Comparison of allergenicity among cow, goat, and horse milks using a murine model of atopy.
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      Physicochemical and functional properties of goat milk whey protein and casein obtained during different lactation stages.
      ). Proteins, as key components of milk, and their physicochemical and functional properties have recently received increased attention (
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      Physicochemical and functional properties of goat milk whey protein and casein obtained during different lactation stages.
      ). To investigate milk proteins' physicochemical and functional properties, milk components should be explored. The results may further reveal the mechanism of protein biosynthesis and intercellular transport in the mammary glands (
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      Human milk proteins: An interactomics and updated functional overview.
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      With the development of molecular biology techniques, proteomics approaches provide a powerful tool for mapping protein profiles and investigating changes in colostrum and mature milk proteins (
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      Changes in whey proteome with lactation stage and parity in dairy cows using a label-free proteomics approach.
      ). For example, several previous studies have outlined the changes in whey and milk fat globule membrane (MFGM) proteins during the lactation stages in human milk (
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      Personalized profiling reveals donor-and lactation-specific trends in the human milk proteome and peptidome.
      ), as well as the differences in whey proteomes from colostrum to mature milk in bovine milk (
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      Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.
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      Bovine milk comparative proteome analysis from early, mid, and late lactation in the cattle breed, Malnad Gidda (Bos indicus).
      ). In addition, MFGM proteins from colostrum to mature milk in goats showed 189 proteins with significant differences, with the acute-phase proteins higher in colostrum than in mature milk (
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      Comparative proteomics of milk fat globule membrane in goat colostrum and mature milk.
      ). Subsequently, MFGM and whey from colostrum and mature goat milk were analyzed using LC-MS proteomics technology (
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      Proteomic analysis of whey proteins in the colostrum and mature milk of Xinong Saanen goats.
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      Characterization of the milk fat globule membrane proteome in colostrum and mature milk of Xinong Saanen goats.
      ). In another study, 2-dimensional gel electrophoresis MALDI-TOF MS technologies compared whey proteins from the first 56 d of lactation in Hu sheep. It was observed that 25 proteins were highly abundant in the first 7 d after lambing, and the expression level decreased to a minimum value at 56 d (
      • Zhang X.
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      The differential composition of whey proteomes in Hu sheep colostrum and milk during different lactation periods.
      ). However, the milk proteome during the lactation stage of dairy goats has received relatively little attention. It is worth noting that the proteome distribution is differently abundant in humans, cattle, camel, yak, and goat milk (
      • Yang Y.
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      Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: Quantitative differential expression patterns.
      ). It is important to explore the distribution, variation, and protein biosynthesis of the mammary glands of the milk proteome of dairy goats over a complete lactation cycle.
      Recently, data-independent acquisition (DIA)-based proteomics has emerged as an alternative to data-dependent acquisition (DDA) in shotgun proteomics (
      • Zhang F.
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      Data-independent acquisition mass spectrometry-based proteomics and software tools: A glimpse in 2020.
      ). Data-independent acquisition parallelizes the fragmentation of all detectable ions within a wide mass/charge (m/z) range, regardless of the intensity, thereby providing a broader dynamic range of the detected signals. This technology has improved the identification, reproducibility, sensitivity, and accuracy of such testing, potentially enhancing proteome-coverage capabilities (
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      ). Data-independent acquisition-based proteomics has been widely applied to characterize protein components in blood, cells, and milk samples (
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      DIA proteomics reveals hypotensive and immune-enhancing constituents in buffalo whey from different altitudes.
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      ). For example, DIA-based proteomics has been used to investigate whey proteins from colostrum to mature milk in humans. Several proteins related to lactation stages, such as plasminogen, lactoferrin, and apolipoprotein A-IV have been identified (
      • Jin D.
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      Comparative analysis of whey proteins in human milk using a data-independent acquisition proteomics approach during the lactation period.
      ).
      Therefore, we hypothesized that proteomic characterization based on the DIA strategy would provide in-depth knowledge of the whey proteome throughout the lactation cycle of dairy goats. This study investigated the whey proteome of goat milk at 1, 3, 30, 90, 150, and 240 d using DIA- and DDA-based quantitative proteomics approaches. The results of this study contribute to enlarging the goat milk protein database, revealing the temporal changes in the goat whey proteome, and exploring the protein biosynthesis and transport of the mammary gland during various lactation stages.

      MATERIALS AND METHODS

      Sample Collection

      The samples were collected from the Qingdao Aote Goat Farm of China. Fifteen healthy dairy goats with second parity and without clinical diseases were selected. The diet ingredients and components of goats during the lactation stages are listed in Supplemental Table S1 (https://doi.org/10.17632/n8n4s2sv9r.3;
      • Sun X.
      Whey preteomics 2021.1.10. Mendeley Data, V3.
      ). At 6 stages (1, 3, 30, 90, 150, and 240 d), 50 mL of milk was collected from the 15 goats. Eighty-six samples were obtained in total; samples from 4 goats were not collected due to insufficient milk on d 240. The dairy goats were housed freely, fed TMR diets twice daily at 0700 and 1630 h for ad libitum intake, and milked twice daily at 0600 and 1800 h. Samples on d 1 and 3 were obtained via manual milking, while the remaining samples were obtained via milking from both udders with a machine. After collection, the samples were stored at −20°C, transferred to the laboratory, and stored at −80°C. Only routine animal procedures (milking) were conducted in this study, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.

      Separation of Milk Whey Proteins

      After the milk samples were thawed at 4°C, 5 individual milk samples from each lactation stage were combined into 1 fraction. This resulted in 3 biological replicates for each lactation stage and 18 pooled samples for the 6-lactation time point. The whole milk was first centrifuged at 4,000 × g and 4°C for 30 min. The top layer of milk fat was removed, and the liquid phase of skim milk was collected. The skim milk was then ultracentrifuged at 100,000 × g for 60 min at 4°C to separate the casein precipitates and the whey protein supernatant using the model L-80XP (Beckman Coulter). The supernatant containing whey proteins was collected and stored at −80°C. Protein concentrations in milk whey were determined using a BCA assay in which bovine serum albumin served as a standard.

      Polyacrylamide Gel Electrophoresis

      A 12% separation gel and a 5% concentration gel were prepared. Twenty micrograms of protein samples were mixed with loading buffer and placed in a 95°C water bath for 5 min. After the samples cooled, the sample and protein marker with 14.4–97.4 kDa (Solarbio) were loaded and electrophoresed. Electrophoresis was performed at 80 V for 20 min and then at 120 V for 60 min. Next, the gel was placed on a plate, fixed with 40% methanol and 10% ethanol, and stained with Coomassie Brilliant Blue G-250 solution. Finally, the gels were incubated in distilled water until the background was colorless and imaged (Supplemental Figure S1; https://doi.org/10.17632/n8n4s2sv9r.3;
      • Sun X.
      Whey preteomics 2021.1.10. Mendeley Data, V3.
      ).

      Protein Digestion

      Thirty micrograms of whey proteins from each lactation time point were heated in 50°C water for 30 min with 30 mM Tris/HCl and 100 mM dithiothreitol. After the samples were cooled, they were mixed with 200 μL of UT buffer (8 M urea and 100 mM Tris-HCl, pH 8.5), transferred into a filter tube (Sartorius), and centrifuged at 14,000 × g and 25°C for 25 min. Subsequently, the samples were washed with UT buffer, mixed with 100 μL of 50 mM iodoacetamide solution, and then incubated for 45 min in the dark at 25°C. After incubation, samples were centrifuged and washed. Finally, the samples were mixed with 100 μL of trypsin buffer (1 μg sequencing grade trypsin in 50 mM NH4HCO3) and then incubated for 16 to 18 h at 37°C. Formic acid (FA) was added to stop the reaction. The filter was transferred to a new tube, centrifuged at 14,000 × g for 15 min, and washed twice with 50 mM NH4HCO3. The eluates containing the peptides were pooled and desalted using a C18 column (60108–303, Thermo Fisher Scientific). The samples were dried in a speed vacuum and stored at −80°C.

      Data-Dependent Acquisition and Data-Independent Acquisition Analysis

      Dried tryptic peptides were resuspended in 0.1% FA and subjected to EASY-nLC 1,000 coupled with Orbitrap Fusion Lumos (Thermo Fisher Scientific). The column was equilibrated with buffer A (0.1% FA). Peptides were loaded onto a C18 trap column (100 μm × 20 mm, 5 μm; Thermo Fisher Scientific) using an autosampler and separated on a C18 analytical column (75 μm × 150 mm, 3 μm; Thermo Fisher Scientific) at a flow rate of 300 nL/min. The separation gradient was as follows: buffer B (80% acetonitrile and 0.1% FA) from 4% to 10% within 5 min, from 10% to 30% within 58 min, from 30% to 40% within 9 min, from 40% to 100% within 8 min, and then 100% hold for 10 min.
      For DDA analysis, MS was performed in positive ion mode with a parent ion scanning range of 300 to 1,800 m/z and automatic switching between MS and MS/MS acquisition. The parameters of MS were set as follows: (1) MS: resolution = 60,000, automatic gain control (AGC) target = 400,000, maximum injection time = 50 ms, and exclusion duration = 40 s. The top 20 most abundant precursor ions with a charge ≥2 from the MS scan were selected and fragmented by higher energy collisional dissociation with normalized collision energies of 27 eV. (2) high energy collisional dissociation (HCD)-MS/MS: resolution = 15,000, AGC target = 50,000, and maximum injection time = 50 ms.
      For DIA analysis, MS was performed in positive ion mode with a parent ion scanning range of 395–1,205 m/z. The parameters of MS were set as follows: (1) MS: resolution = 60,000, AGC target = 2 × 106, and maximum injection time = 100 ms; (2) HCD-MS/MS: resolution = 15,000, AGC target = 1 × 106, and collision energy = 30 eV; (3) a DIA using an isolation width of 26 Da (containing 1 Da for the window overlap) and 32 overlapping windows were constructed covering the precursor mass range of 400 to 1,200 Da for DIA acquisition.

      Protein Identification and Quantification

      The DDA raw files were analyzed using MaxQuant software (version 2.0.3.0) to search against the database downloaded from UniProt (46,754 entries of Bos taurus; 35,479 entries of Capra hircus; downloaded in December 2020). The relevant parameters were set as follows: the digestion mode was set to trypsin/P specificity, maximum missed cleavages at 2, fixed carbamidomethyl modification of cysteine, and variable modifications of N-terminal acetylation and methionine oxidation. Protein and peptide identifications were achieved at a false discovery rate and Peptide-Spectrum matching of 0.01. The conditions for matching between runs were set as 0.7 match time window, 0.05 ion mobility, 20 alignment time windows, and 1 alignment ion mobility. The identified proteins were quantified based on the abundance of razor and unique peptides using a label-free quantitation (LFQ) workflow. The DIA raw files were also searched against the downloaded database using the MaxQuant software, as mentioned above. In addition, the spectral library was established using DDA, and the other parameter settings were the same as those applied in the DDA procedure.

      Bioinformatics and Statistical Analysis

      The whey proteins with at least 2 identified peptides and all 3 runs of each studied group were selected and imported into the Perseus software (www.maxquant.org/perseus/). Hierarchical clustering, volcano plots, principal component analysis (PCA), and statistical analysis of the quantified proteins among the studied groups were performed. Quantified proteins among the studied groups were analyzed using ANOVA with Benjamini-Hochberg false discovery rate. Differentially abundant proteins were determined according to |fold-change| ≥2 and q-value <0.05. Gene Ontology (GO) enrichment and the Kyoto Encyclopedia of Gene and Genome (KEGG) pathway of these different proteins were analyzed using DAVID Bioinformatics Resources 6.8 software (david.ncifcrf.gov/summary.jsp), in which P-value <0.05 was considered to be significantly enriched. The protein-protein interactions (PPI) of differentially abundant proteins were predicted using STRING software (string-db.org) with 0.70 confidence and visualized using Cytoscape software.

      RESULTS

      Identification and Quantification of Whey Proteins

      Clustering analysis of the quantified whey proteins in goat milk collected during the entire lactation cycle is shown in Supplemental Figure S2 (https://doi.org/10.17632/n8n4s2sv9r.3;
      • Sun X.
      Whey preteomics 2021.1.10. Mendeley Data, V3.
      ). Using the DIA proteomics method, 344 proteins were identified in the whey component of goat milk throughout the lactation cycle (Supplemental Table S2; https://doi.org/10.17632/n8n4s2sv9r.3;
      • Sun X.
      Whey preteomics 2021.1.10. Mendeley Data, V3.
      ). We found that the samples from d 90, 150, and 240 formed a subcluster, samples from d 30 joined them and formed a large cluster, and samples from d 1 and 3 formed another subcluster and then a cluster. Using the DDA proteomics method, 331 whey proteins were identified in goat milk throughout the lactation cycle (Supplemental Table S3; https://doi.org/10.17632/n8n4s2sv9r.3;
      • Sun X.
      Whey preteomics 2021.1.10. Mendeley Data, V3.
      ). The clustering analysis results using the approach based on quantified whey proteins and DDA proteomics were consistent with those of the DIA proteomics approach. The whey proteomes in dairy goats at d 90, 150, and 240 were more similar to one another than those at d 30, whereas the whey proteomes at d 1 and d 3 were similar.
      The PCA of the quantified whey proteins from the 6 stages in the goat milk cycle using the DIA method is presented in Figure 1a. According to the score plots, the protein profiles of the 1-, 3-, 30-, and 240-d whey components were clustered, whereas the whey protein profiles at d 90 and 150 were not distinguishable from one another. Apparent changes in the whey proteome profile throughout the lactation stages were revealed, and there were apparent differences in the proteomes in the 1-, 3-, 30-, 90-, 150-, and 240-d groups. Principal components 1 and 2 accounted for 52.6% of the total variance at the different lactation stages. The score plots of the PCA based on the DDA data are shown in Figure 1b. We found that apparent changes in the whey proteome profiles throughout the lactation stages in the DDA data were similar to those in the DIA data.
      Figure thumbnail gr1
      Figure 1Principal components analysis of whey proteins from 1, 3, 30, 90, 150, and 240 d of dairy goats using (a) data-independent acquisition (DIA) and (b) data-dependent acquisition (DDA)-based proteomics approaches.

      Differentially Abundant Whey Proteins Throughout the Lactation Stages

      For the DIA data, 238 proteins were considerably different throughout the lactation stages according to q-value and fold-change (Supplemental Table S4; https://doi.org/10.17632/n8n4s2sv9r.3;
      • Sun X.
      Whey preteomics 2021.1.10. Mendeley Data, V3.
      ). Several proteins, including IgG, lipopolysaccharide-binding protein (LBP), fibronectin, serotransferrin, melanotransferrin, inter-α-trypsin inhibitor, α-1-antiproteinase, prothrombin, α-2 macroglobulin, complement C3, and growth/differentiation factor 8 were significantly decreased from d 1 to 240. Lipoprotein lipase, fatty acid-binding protein 3 (FABP3), lactoperoxidase, polymeric immunoglobulin receptor (PIGR), osteopontin, and endoplasmin were significantly increased from d 1 to 240. In addition, lactoferrin, actin-depolymerizing factor, and cysteine-rich secretory protein 3 levels decreased from d 1 to 30 and then increased until d 240.
      To visualize the changes in whey proteins during the lactation stages, volcano plots comparing d 1 and d 3, 30, 90, 150, and 240 are shown in Figure 2 and Supplemental Table S5 (https://doi.org/10.17632/n8n4s2sv9r.3;
      • Sun X.
      Whey preteomics 2021.1.10. Mendeley Data, V3.
      ). Compared with the d 1 sample, significantly altered proteins increased from d 3 to d 90 and remained similar to those at d 240. Changes in the components of whey protein profiles in goat milk increased from d 1 to 90 during the lactation stage. Of these, we found that the levels of lactoperoxidase, syndecan, and FABP3 were significantly increased, whereas those of complement C3, complement subcomponent C1r, α-2 macroglobulin, matrix Gla protein, and IgG were significantly decreased from d 1 to 240. In addition, the changes in the abundance of most whey proteins in goat milk throughout the lactation stages from the DDA data were consistent with those from the DIA data. Several of the differentially abundant proteins from the studied groups, such as apolipoprotein A-IV, lactoperoxidase, FABP3, fibrinogen, complement C3, and vitamin D-binding protein, are shown in Figures 3a and 3b.
      Figure thumbnail gr2
      Figure 2Volcano plots of differentially abundant whey proteins from 3 d versus 1 d, 30 d versus 1 d, 90 d versus 1 d, 150 d versus 1 d, and 240 d versus 1 d of dairy goats using data-independent acquisition (DIA)-based proteomics approach. FC = fold change.
      Figure thumbnail gr3
      Figure 3Clustering analysis of representative differentially abundant whey proteins from 1, 3, 30, 90, 150, and 240 d of dairy goats using (a) data-independent acquisition (DIA) and (b) data-dependent acquisition (DDA)-based proteomics approaches.

      Bioinformatics Analysis of Differentially Abundant Whey Proteins

      According to the protein annotations, the 238 differentially abundant whey proteins identified in goat milk were classified according to biological process, cellular component, and molecular function. As shown in Figure 4, most biological processes were a response to stimulus, localization, protein metabolic process, transport, and immune system processes. The most abundant cellular components were the extracellular region and membrane-bound vesicle, and other proteins were located in the extracellular vesicle, exosome, and extracellular space. The most common molecular functions of differential proteins are protein binding, carbohydrate derivative binding, molecular function regulation, and receptor binding. We also found that the number of differentially abundant proteins involved in most GO terms increased when comparing d 1 and 3 to d 1 and 240.
      Figure thumbnail gr4
      Figure 4(a) Biological processes, (b) cellular component, and (c) molecular functions of differentially abundant whey proteins among the comparisons of 3 d versus 1 d, 30 d versus 1 d, 90 d versus 1 d, 150 d versus 1 d, and 240 d versus 1 d of dairy goats using data-independent acquisition (DIA)-based proteomics approach.
      The protein networks of the differentially abundant whey proteins identified in goat milk throughout the lactation cycle were predicted using STRING software and visualized using Cytoscape software (Figure 5). Active PPI sources from experiments, curated databases, and text mining were selected, and the minimum required interaction score was set at high confidence (0.70). In the protein network comparing d 1 and 3, fructose-bisphosphate aldolase with a higher degree of connectivity was considered a hub node. In the network comparing d 1 and 30, fibronectin with a higher degree of connectivity was considered a hub node. In comparing d 1 and 90, endoplasmin, α-1-antiproteinase, and fibronectin with a higher degree of connectivity were considered central hub nodes. In comparing d 1 and 150, fibronectin, endoplasmin, α-1-antiproteinase, and endoplasmic reticulum chaperone BiP were considered the central hub nodes. In the comparison protein network of d 1 and 240, fibronectin and α-1-antiproteinase were considered central hub nodes. Collectively, fibronectin, endoplasmin, and α-1-antiproteinase were considered the central proteins among the comparisons at d 1 and 90, 1 and 150, and 1 and 240.
      Figure thumbnail gr5
      Figure 5Protein-protein interactions (PPI) of differentially abundant whey proteins from (a) 3 d versus 1 d, (b) 30 d versus 1 d, (c) 90 d versus 1 d, (d) 150 d versus 1 d, and (e) 240 d versus 1 d of dairy goats using data-independent acquisition (DIA)-based proteomics approach. Each node represents a protein, and each edge represents the interaction between proteins. Pink box means upregulated proteins. Blue box means downregulated proteins.
      Differentially abundant whey proteins were classified into KEGG pathways (Table 1). The results showed that most of the differentially abundant whey proteins were involved in the complement and coagulation cascades, regulation of actin cytoskeleton, Staphylococcus aureus infection, and phagosomes. In addition, several differentially abundant proteins, such as PIGR, fructose-bisphosphate aldolase, and connective tissue growth factor (CTGF), were related to the biosynthesis of antibiotics, AA, antigen processing and presentation, and Hippo signaling pathways.
      Table 1Pathway analysis of differentially abundant whey proteins from 1, 3, 30, 90, 150, and 240 d of dairy goats using data-independent acquisition (DIA)-based proteomics approach
      TermCountHitsPercentageP-valueFold enrichment
      3 d vs. 1 d
       Biosynthesis of antibiotics620611.321.37E-036.87
       Glycolysis/gluconeogenesis5639.431.17E-0418.73
       Biosynthesis of AA5719.431.87E-0416.62
      Salmonella infection5839.433.41E-0414.21
       Carbon metabolism51099.439.58E-0410.82
       Phagosome51589.433.74E-037.47
       Regulation of actin cytoskeleton52129.431.05E-025.56
       Hippo signaling pathway41517.552.34E-026.25
       PPAR signaling pathway3705.663.31E-0210.11
      30 d vs. 1 d
       Protein processing in endoplasmic reticulum816910.534.06E-058.12
       Antigen processing and presentation7759.213.57E-0616.02
       Phagosome51586.581.21E-025.43
       Proteoglycans in cancer52036.582.76E-024.23
       Biosynthesis of antibiotics52066.582.90E-024.16
       Biosynthesis of AA4715.267.51E-039.67
       Estrogen signaling pathway4985.261.80E-027.00
       Carbon metabolism41095.262.38E-026.30
       Legionellosis3573.954.15E-029.03
      90 d vs. 1 d
       Regulation of actin cytoskeleton82128.518.05E-045.09
       Biosynthesis of antibiotics82068.516.79E-045.24
       Biosynthesis of AA7717.451.13E-0513.29
       Carbon metabolism71097.451.29E-048.66
       Phagosome71587.459.49E-045.97
       Protein processing in endoplasmic reticulum71697.451.35E-035.58
       PI3K-Akt signaling pathway73477.453.93E-022.72
       Glycolysis/gluconeogenesis6636.388.69E-0512.84
       Complement and coagulation cascades6746.381.87E-0410.93
       Hippo signaling pathway61516.384.66E-035.36
       Proteoglycans in cancer62036.381.57E-023.98
       Antigen processing and presentation5755.322.09E-038.99
      Salmonella infection5835.323.03E-038.12
       Tuberculosis51815.324.23E-023.72
       Legionellosis4574.268.11E-039.46
       Bacterial invasion of epithelial cells4774.261.83E-027.00
       Pertussis4774.261.83E-027.00
       Estrogen signaling pathway4984.263.42E-025.50
       Amoebiasis41124.264.78E-024.82
      150 d vs. 1 d
       Regulation of actin cytoskeleton82128.425.70E-045.38
       Complement and coagulation cascades7747.371.03E-0513.48
       Protein processing in endoplasmic reticulum71697.379.99E-045.90
       Proteoglycans in cancer62036.321.25E-024.21
       Biosynthesis of antibiotics62066.321.32E-024.15
       Biosynthesis of AA5715.261.38E-0310.03
       Antigen processing and presentation5755.261.70E-039.50
      Salmonella infection5835.262.46E-038.58
       Carbon metabolism51095.266.54E-036.53
       Hippo signaling pathway51515.261.98E-024.72
       Phagosome51585.262.30E-024.51
       Glycolysis/gluconeogenesis4634.219.15E-039.04
       Bacterial invasion of epithelial cells4774.211.58E-027.40
       Pertussis4774.211.58E-027.40
       Estrogen signaling pathway4984.212.96E-025.81
       Amoebiasis41124.214.15E-025.09
      240 d vs. 1 d
       Regulation of actin cytoskeleton92128.331.71E-045.53
       Biosynthesis of antibiotics82067.418.45E-045.06
       Biosynthesis of AA7716.481.39E-0512.83
       Complement and coagulation cascades7746.481.76E-0512.31
       Carbon metabolism71096.481.57E-048.36
       Hippo signaling pathway71516.489.07E-046.03
       Phagosome71586.481.15E-035.77
       Viral carcinogenesis72346.488.06E-033.89
       PI3K-Akt signaling pathway73476.484.57E-022.63
       Glycolysis/gluconeogenesis6635.561.03E-0412.40
      Salmonella infection6835.563.80E-049.41
       Protein processing in endoplasmic reticulum61695.568.67E-034.62
       Proteoglycans in cancer62035.561.81E-023.85
       Antigen processing and presentation5754.632.38E-038.68
       Bacterial invasion of epithelial cells5774.632.62E-038.45
       Tuberculosis51814.634.72E-023.60
       Legionellosis4573.708.95E-039.13
       Pertussis4773.702.01E-026.76

      DISCUSSION

      Characterization of the Identified Whey Proteins

      Previous studies utilized LC-MS/MS proteomics technology to investigate goat colostrum and mature whey, wherein 314 and 524 whey proteins were identified in goat colostrum and mature milk, respectively (
      • Sun Y.
      • Wang C.
      • Sun X.
      • Guo M.
      Proteomic analysis of whey proteins in the colostrum and mature milk of Xinong Saanen goats.
      ). Another study that utilized Q-Orbitrap high resolution mass spectrometry (HRMS) analysis expanded the proteome to 400 whey proteins in goat colostrum and mature milk (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ). EASY-nLC-Orbitrap LTQ VELOS detected MFGM proteins from colostrum and mature milk in goat milk. A total of 423 proteins were identified, 189 of which were significantly different (
      • Lu J.
      • Liu L.
      • Pang X.
      • Zhang S.
      • Jia Z.
      • Ma C.
      • Zhao L.
      • Lv J.
      Comparative proteomics of milk fat globule membrane in goat colostrum and mature milk.
      ). Compared with previous studies, changes in whey proteins in goat milk throughout the lactation cycle were first investigated using a DIA-based proteomics approach that elucidated the main results of this study, whereas the results of the DDA proteomics method can be used to verify the accuracy of the DIA results. Among these, 92 proteins including IgG, melanotransferrin, apolipoprotein E, α-1-antiproteinase, and fibronectin decreased from d 1 to 240, whereas 85 proteins including lactoperoxidase, PIGR, and osteopontin increased from d 1 to 240. In addition, several differentially abundant proteins were involved in immune system processes, complement and coagulation cascades, protease inhibition, and transport.

      Differentially Abundant Proteins Associated with Immune Function

      According to GO analysis, several differentially abundant proteins involved in immune system processes, such as α-2-macroglobulin, immunoglobulin, peptidoglycan-recognition protein, LBP, and melanotransferrin were identified, and these proteins decreased from d 1 to 240. In a previous study, IgG was found to be higher in colostrum than in mature bovine and human milk using iTRAQ labeling proteomics (
      • Yang M.
      • Cong M.
      • Peng X.
      • Wu J.
      • Wu R.
      • Liu B.
      • Ye W.
      • Yue X.
      Quantitative proteomic analysis of milk fat globule membrane (MFGM) proteins in human and bovine colostrum and mature milk samples through iTRAQ labeling.
      ). In small ruminants' milk, IgG was highly abundant at 0 d and then declined, reaching the lowest level at 56 d in sheep milk using 2-dimensional gel electrophoresis and MALDI-TOF MS technologies (
      • Zhang X.
      • Liu X.
      • Li F.
      • Yue X.
      The differential composition of whey proteomes in Hu sheep colostrum and milk during different lactation periods.
      ). In nature, IgG is the highest in the maternal colostrum, which corresponds to the intestinal closure of neonatal ruminants. This result is related to lambs' reliance on the timely ingestion of IgG via colostrum to acquire an initial passive immunity that protects them against the invasion of various pathogens during early life (
      • Moore R.E.
      • Townsend S.D.
      Temporal development of the infant gut microbiome.
      ). According to other immune-related proteins, α-2-macroglobulin can eliminate endogenous and exogenous proteases produced by invading pathogens and parasites, thereby acting as a humoral defense barrier against pathogens with a unique pan-protease inhibitor function (
      • Rehman A.A.
      • Ahsan H.
      • Khan F.H.
      Alpha-2-macroglobulin: A physiological guardian.
      ). Alpha-2-macroglobulin has been found to be approximately 7-fold higher in colostrum than in mature milk in goat whey using LC-MS/MS proteomics technology (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ). In goat MFGM, α-2-macroglobulin levels were approximately 4-fold higher in colostrum than in mature milk using a label-free proteomics approach (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      A high-throughput comparative proteomics of milk fat globule membrane reveals breed and lactation stages specific variation in protein abundance and functional differences between milk of Saanen dairy goat and Holstein bovine.
      ). In addition to playing a protective role, immune proteins can bind to receptors on intestinal epithelial cells to activate downstream signals. Lipopolysaccharide-binding protein can bind to bacterial lipopolysaccharides to initiate an immune response and catalyze the transfer of bacterial lipopolysaccharide to CD14. Interactions between CD14 and LBP are necessary to activate toll-like receptors 2 and 4, followed by the activation of signal transduction pathways and the production of cytokines in response to LPS (
      • Meng L.
      • Song Z.
      • Liu A.
      • Dahmen U.
      • Yang X.
      • Fang H.
      Effects of lipopolysaccharide-binding protein (LBP) single nucleotide polymorphism (SNP) in infections, inflammatory diseases, metabolic disorders and cancers.
      ). Lipopolysaccharide-binding protein was found in goat colostrum using Q-Orbitrap HRMS proteomics technology (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ). The LBP concentration during early lactation (63.3 μg/mL) significantly decreased to 27.5 μg/mL during late lactation in bovine milk when analyzed using an ELISA (
      • Wenz J.R.
      • Fox L.
      • Muller F.
      • Rinaldi M.
      • Zeng R.
      • Bannerman D.
      Factors associated with concentrations of select cytokine and acute phase proteins in dairy cows with naturally occurring clinical mastitis.
      ). In the goat MFGM fraction, LBP in colostrum was approximately 3-fold higher than that in mature milk as observed using LFQ-based proteomics. This observation was confirmed using ELISA (
      • Lu J.
      • Liu L.
      • Pang X.
      • Zhang S.
      • Jia Z.
      • Ma C.
      • Zhao L.
      • Lv J.
      Comparative proteomics of milk fat globule membrane in goat colostrum and mature milk.
      ). As previously discussed, the high abundance of LBP in colostrum helps neonates protect themselves from bacterial invasion. In addition, milk has considerable potential as a source of high-quality protein to produce healthy food products.
      Several proteins, such as PIGR and lactoperoxidase, increased from d 1 to 240. Lactoperoxidase is one of the most prominent enzymes and is a component of natural antimicrobial systems in raw milk. Lactoperoxidase tended to increase from 0.5 to 2 mo and then decrease from 9 to 12 mo in bovine milk using filter-aided sample preparation combined with a dimethyl labeling proteomics approach (
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.
      ).
      • Mol P.
      • Kannegundla U.
      • Dey G.
      • Gopalakrishnan L.
      • Dammalli M.
      • Kumar M.
      • Patil A.H.
      • Basavaraju M.
      • Rao A.
      • Ramesha K.P.
      • Prasad T.S.K.
      Bovine milk comparative proteome analysis from early, mid, and late lactation in the cattle breed, Malnad Gidda (Bos indicus).
      used tandem mass tag labeling proteome technology and found that lactoperoxidase decreased from early to late lactation in indigenous Indian cattle. Our results are partly consistent with those of Zhang's group (
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Bovine milk proteome in the first 9 days: Protein interactions in maturation of the immune and digestive system of the newborn.
      ). This observation could be related to the intrinsic characteristics of goat milk, wherein the lactoperoxidase system catalyzes the oxidation of thiocyanate to antibacterial hypothiocyanite and contributes to the conservation of the lactating mammary gland during involution (
      • Zou Z.
      • Bauland J.
      • Hewavitharana A.K.
      • Al-Shehri S.S.
      • Duley J.A.
      • Cowley D.M.
      • Koorts P.
      • Shaw P.N.
      • Bansal N.
      A sensitive, high-throughput fluorescent method for the determination of lactoperoxidase activities in milk and comparison in human, bovine, goat and camel milk.
      ). Polymeric immunoglobulin receptors contribute to bridging the innate and adaptive immune responses at mucosal surfaces. In dairy goats, PIGR was approximately 133-fold higher in colostrum than in the whey fraction of mature milk, and 26-fold higher in colostrum than in the MFGM fraction of mature milk using Q-Orbitrap HRMS-based proteomics techniques (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ,
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      A high-throughput comparative proteomics of milk fat globule membrane reveals breed and lactation stages specific variation in protein abundance and functional differences between milk of Saanen dairy goat and Holstein bovine.
      ). It was lower in colostrum than in mature milk in yaks using iTRAQ-labeled proteomics (
      • Yang Y.
      • Zhao X.
      • Yu S.
      • Cao S.
      Quantitative proteomic analysis of whey proteins in the colostrum and mature milk of yak (Bos grunniens).
      ).
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.
      used filter-aided sample preparation combined with dimethyl labeling followed by LC-MS/MS and found that PIGR increased from 0.5 to 12 mo in bovine milk. As discussed previously, we speculated that PIGR with high abundance in the late lactation stage of ruminant dairy animals might be related to the mammary gland immune response by transporting polymeric immunoglobulins, such as IgA and IgM, across mucosal epithelial cells (
      • Matsumoto M.L.
      Molecular mechanisms of multimeric assembly of IgM and IgA.
      ).
      In our study, lactoferrin decreased from d 1 to 30 and then increased until d 240. Lactoferrin was approximately 51-fold higher in colostrum than in mature whey of goat (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ). A previous study found that lactoferrin in bovine milk was low at 0.5 mo and increased as lactation advanced (
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.
      ). Lactoferrin was significantly influenced by lactation stages, with correlation coefficients of 0.557 that increased from early to late lactation in bovine milk. The late lactation with the highest lactoferrin concentration of 156.68 μg/mL was found by
      • Cheng J.B.
      • Wang J.Q.
      • Bu D.P.
      • Liu G.L.
      • Zhang C.G.
      • Wei H.Y.
      • Zhou L.Y.
      • Wang J.Z.
      Factors affecting the lactoferrin concentration in bovine milk.
      using the ELISA method. In goat milk, lactoferrin was highest in the colostrum (387 ± 69 μg/mL), rapidly decreased in the following week (62 ± 25 μg/mL), and then increased in late lactation (107 ± 19 μg/mL;
      • Hiss S.
      • Meyer T.
      • Sauerwein H.
      Lactoferrin concentrations in goat milk throughout lactation.
      ). Our results are similar to those of Hiss's group. As discussed previously, the changes in LBP, lactoperoxidase, PIGR, and lactoferrin during the lactation stage may be related to their protective roles in neonatal and goat mammary glands against infections during involution.

      Differentially Abundant Proteins Related to Complement and Coagulation Cascades

      We found that several proteins participate in complement and coagulation cascades, including complement C3, complement subcomponent C1r, fibronectin, and complement C5, which decreased from d 1 to 240. A previous study found that complement C3 in colostrum was higher than in mature human and bovine milk using an iTRAQ labeling proteomic method (
      • Yang M.
      • Cao X.
      • Wu R.
      • Liu B.
      • Ye W.
      • Yue X.
      • Wu J.
      Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.
      ). In goat milk, complement C3 was also approximately 3-fold higher in colostrum than in the whey fraction of mature milk (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ). This observation may be related to complement C3 serving a central role in the complement system, in which processing is the central response in the classical and alternative pathways. A deficiency of complement C3 can lead to susceptibility to bacterial infections (
      • Sorbara M.T.
      • Foerster E.G.
      • Tsalikis J.
      • Abdel-Nour M.
      • Mangiapane J.
      • Sirluck-Schroeder I.
      • Tattoli I.
      • van Dalen R.
      • Isenman D.E.
      • Rohde J.R.
      • Girardin S.E.
      • Philpott D.J.
      Complement C3 drives autophagy-dependent restriction of cyto-invasive bacteria.
      ).
      Fibronectin was found in Saanen goat milk using DIA proteomics technology (
      • Zhao Z.
      • Liu N.
      • Wang C.
      • Cheng J.
      • Guo M.
      Proteomic analysis of differentially expressed whey proteins in Saanen goat milk from different provinces in China using a data-independent acquisition technique.
      ) and decreased from d 1 to 9 in bovine milk using dimethyl labeling proteomic technology (
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Bovine milk proteome in the first 9 days: Protein interactions in maturation of the immune and digestive system of the newborn.
      ). In another study, fibronectin was only identified in goat colostrum and was not detected in mature milk based on an LC-MS/MS proteomics approach (
      • Sun Y.
      • Wang C.
      • Sun X.
      • Guo M.
      Proteomic analysis of whey proteins in the colostrum and mature milk of Xinong Saanen goats.
      ). Fibronectin is an acute-phase reactive protein that protects the host from bacterial infections (
      • Orczyk-Pawiłowicz M.
      • Hirnle L.
      • Berghausen-Mazur M.
      • Kątnik-Prastowska I.
      Terminal glycotope expression on milk fibronectin differs from plasma fibronectin and changes over lactation.
      ). In addition, fibronectin mediates cell interactions and plays an essential role in cell adhesion, migration, proliferation, and extracellular matrix remodeling (
      • Zollinger A.J.
      • Smith M.L.
      Fibronectin, the extracellular glue.
      ). We also found that fibronectin, with more interactions than other proteins in PPI, could serve as a crucial protein contributing to the regulation of the complement and coagulation cascades. Complement and coagulation cascades are an essential part of the immune system, and the coexistence and interaction of complement and coagulation cascades in the same microenvironment generally ensures successful host immune defense in an impaired barrier environment (
      • Oikonomopoulou K.
      • Ricklin D.
      • Ward P.A.
      • Lambris J.D.
      Interactions between coagulation and complement—Their role in inflammation.
      ).

      Differentially Abundant Proteins Involved in Protease Activity

      In our study, α-1-antiproteinase, inter-α-trypsin inhibitor, plasminogen, and antithrombin-III levels decreased from d 1 to 240. A previous study found that plasminogen, inter-α-trypsin inhibitor, and antithrombin-III levels were higher in bovine colostrum than in mature milk (
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Bovine milk proteome in the first 9 days: Protein interactions in maturation of the immune and digestive system of the newborn.
      ). In human milk, plasminogen levels were higher in colostrum than in mature milk using a DIA proteomics approach (
      • Jin D.
      • Liu H.
      • Bu L.
      • Ke Q.
      • Li Z.
      • Han W.
      • Zhu S.
      • Liu C.
      Comparative analysis of whey proteins in human milk using a data-independent acquisition proteomics approach during the lactation period.
      ). In goat milk, plasminogen was increased by approximately 3-fold in the whey fraction of colostrum compared with that in mature milk (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ). Plasminogens can be converted into active plasmin by plasminogen activators. The plasmin system can interact with other milk components such as whey and casein proteins and promote proteolysis (
      • Ismail B.
      • Nielsen S.
      Invited review: Plasmin protease in milk: Current knowledge and relevance to dairy industry.
      ). According to α-1-antiproteinase, it was decreased with prolonged lactation in human milk based on dimethyl labeling and LFQ proteomics approaches (
      • Liao Y.
      • Alvarado R.
      • Phinney B.
      • Lönnerdal B.
      Proteomic characterization of human milk whey proteins during a twelve-month lactation period.
      ;
      • Zhang L.
      • de Waard M.
      • Verheijen H.
      • Boeren S.
      • Hageman J.A.
      • Van Hooijdonk T.
      • Vervoort J.
      • Van Goudoever J.B.
      • Hettinga K.
      Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.
      ). Alpha-1-antiproteinase was also higher in colostrum than in mature milk in humans and bovines using an iTRAQ-based proteomics approach (
      • Yang M.
      • Cao X.
      • Wu R.
      • Liu B.
      • Ye W.
      • Yue X.
      • Wu J.
      Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.
      ). In goat milk, α-1-antiproteinase was increased by approximately 13-fold in the whey fraction of colostrum compared with that of mature milk (
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      ). Our results are similar to the results of above previous studies. The higher abundance of α-1-antiproteinase may contribute to the protection of immune-related proteins such as LBP, lactoferrin, and IgG against proteolysis, especially IgG, across the intestine in neonatal calves (
      • Wang X.X.
      • Han R.W.
      • Zhao X.W.
      • Huang D.W.
      • Zhu H.L.
      • Wu T.
      • Qi Y.X.
      • Yang Y.X.
      • Cheng G.L.
      Label-free quantitative proteomics analysis reveals the fate of colostrum proteins in the intestine of neonatal calves.
      ). Our results revealed that α-1-antiproteinase and plasminogen interact with several proteins, such as hemopexin, fibronectin, and CD59 glycoprotein, and may inhibit proteolysis to maintain milk protein stability. Serpins protect cells, resist proteases, and play an important role in regulating the proteolysis process (
      • Spence M.A.
      • Mortimer M.D.
      • Buckle A.M.
      • Minh B.Q.
      • Jackson C.J.
      A comprehensive phylogenetic analysis of the serpin superfamily.
      ). Several previous studies have indicated that serpins A3–5 and A3–7 decreased from colostrum to mature milk in bovines (
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.
      ,
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Bovine milk proteome in the first 9 days: Protein interactions in maturation of the immune and digestive system of the newborn.
      ). Therefore, we speculated that protease inhibitors with high abundance in colostrum might protect immune-related proteins against proteolysis, especially by transporting IgG across the intestine into the blood and promoting the maturation of the immune system in neonates.

      Differentially Abundant Proteins Involved in Transport

      Our study found that several proteins involved in transport, such as FABP3, nucleobindin 2, calgranulin-A, and calgranulin-B, increased from d 1 to 240. It has been reported that calgranulin-A and calgranulin-B are calcium-binding proteins involved in the antimicrobial functions and activation of cytokines and chemokines. However,
      • Honan M.C.
      • Fahey M.J.
      • Fischer-Tlustos A.J.
      • Steele M.A.
      • Greenwood S.L.
      Shifts in the Holstein dairy cow milk fat globule membrane proteome that occur during the first week of lactation are affected by parity.
      found that calgranulin-A and calgranulin-B levels decreased from colostrum to mature milk in bovine MFGM. Our findings were inconsistent with this result. This observation may depend on the type of dairy species and requires further investigation.
      • Zhang Q.
      • Cundiff J.K.
      • Maria S.D.
      • McMahon R.J.
      • Woo J.G.
      • Davidson B.S.
      • Morrow A.L.
      Quantitative analysis of the human milk whey proteome reveals developing milk and mammary-gland functions across the first year of lactation.
      investigated the changes in whey proteome in human milk produced from 1 wk to 12 mo after birth using the tandem mass tag labeled proteomics approach and found that FABP3 levels increased from 1 wk to 12 mo. In bovine,
      • Bionaz M.
      • Hurley W.
      • Loor J.
      Milk protein synthesis in the lactating mammary gland: Insights from transcriptomics analyses.
      found that the expression level of FABP3 in mammary glands increased from 0 to 40 d after delivery using a transcriptomics approach. FABP3, through the uptake and transport of exogenous fatty acids into breast epithelial cells, plays a vital role in breast fatty acid transport (
      • Ye T.
      • Shaukat A.
      • Yang L.
      • Chen C.
      • Zhou Y.
      • Yang L.
      Evolutionary and association analysis of buffalo FABP family genes reveal their potential role in milk performance.
      ).
      In addition, several transport-related proteins, such as hemopexin, vitamin D-binding protein, apolipoprotein A-IV/H/E, and thrombospondin 1, decreased from d 1 to 240. In bovine milk, apolipoprotein E decreased from 0.5 mo to the middle of the lactation cycle (
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.
      ). However, in human milk, apolipoprotein E decreased 30% from 1 wk to 12 mo (
      • Zhang Q.
      • Cundiff J.K.
      • Maria S.D.
      • McMahon R.J.
      • Woo J.G.
      • Davidson B.S.
      • Morrow A.L.
      Quantitative analysis of the human milk whey proteome reveals developing milk and mammary-gland functions across the first year of lactation.
      ). Apolipoprotein E is involved in cholesterol transport. Cholesterol plays a vital role in the synthesis of steroid hormones and vitamin D, which is critical for the development of neonates (
      • Xu L.
      • Shi L.
      • Liu L.
      • Liang R.
      • Li Q.
      • Li J.
      • Han B.
      • Sun D.
      Analysis of liver proteome and identification of critical proteins affecting milk fat, protein and lactose metabolism in dairy cattle with iTRAQ.
      ).
      • Zhang Q.
      • Cundiff J.K.
      • Maria S.D.
      • McMahon R.J.
      • Woo J.G.
      • Davidson B.S.
      • Morrow A.L.
      Quantitative analysis of the human milk whey proteome reveals developing milk and mammary-gland functions across the first year of lactation.
      did not find differences in the vitamin D-binding protein between samples collected at 1 wk and 1, 3, 6, 9, and 12 mo using a tandem mass tag labeling proteomics approach. Using a tandem mass tag labeling proteomics approach,
      • Zhang L.
      • Boeren S.
      • Hageman J.A.
      • van Hooijdonk T.
      • Vervoort J.
      • Hettinga K.
      Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.
      found that in bovine milk, vitamin D-binding protein increased in whey from 0.5 to 3 mo and then decreased until 9 mo. In goat milk,
      • Jia W.
      • Zhang R.
      • Zhu Z.
      • Shi L.
      LC-Q-Orbitrap HRMS-based proteomics reveals potential nutritional function of goat whey fraction.
      found that the vitamin D-binding protein level was approximately 2-fold higher in colostrum than in mature milk whey using HRMS-based proteomics techniques. It is well known that vitamin D-binding protein is primarily responsible for preventing vitamin D from biodegradation. Thus, we speculated that proteins related to nutrient transport could contribute to enhancing the immune system and promoting the development and growth of neonates.

      CONCLUSIONS

      Related proteins corresponding to specific stages of lactation were revealed using a DIA-based proteomics approach that was confirmed using a DDA quantitative proteomics strategy. The proteins IgG, vitamin D-binding protein, LBP, and fibronectin decreased from d 1 to 240, whereas lactoperoxidase, PIGR, calgranulin-A, and calgranulin-B increased from d 1 to 240. Fibronectin with a higher degree of connectivity was considered a central node. These findings provide new insights into the whey proteome profile and temporary changes in whey proteins throughout the lactation cycle, which may contribute to understanding the intrinsic physiological functions of goats.

      ACKNOWLEDGMENTS

      This work was supported by Shandong Provincial Natural Science Foundation (ZR2020MC209; Jinan, China), the School-Land Integration Development Project (2021XDRHXMQT34; Yantai, China), the Qingdao Science and Technology Demonstration and Guidance Project (21-1-4-ny-17-nsh; Qingdao, China), and the Postgraduate Innovation Program of Qingdao Agricultural University (QYNCX20069; Qingdao, China). We thank Kun Xu from the Key Laboratory of Qingdao Agricultural University (Shandong, China) for providing technical support. The authors have not stated any conflicts of interest.

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