Exploration of ovine milk whey proteome during postnatal development using an iTRAQ approach

Introduction

Milk as a complex body fluid synthesized by mammals, contains a large number of secreted proteins that contribute to the growth, development and immune system of neonates (Roncada et al., 2012; Hernandez-Castellano et al., 2015b; Li et al., 2019). Sheep milk contains a wide range of proteins, including the high-abundance proteins α_s1-casein (α_s1-CN), α_s2-casein (α_s2-CN), β-casein (β-CN), κ-casein (κ-CN), β-lactoglobulin (β-Lg) and α-lactalbumin (α-Lac; Gidlund et al., 2015) and low-abundance proteins such as immunoglobulin, lactoferrin, hormones and enzymes (Le et al., 2011). The proteins in mammalian milk can be mainly divided into three classes: caseins (CNs), whey proteins and milk fat globule membrane proteins (MFGMPs). Among these classes, CNs are primary proteins that present in micellar form, whereas whey fractions occur in soluble form (Roncada et al., 2012). MFGMPs are mainly present in the cream portion of milk, which is composed by proteins and lipids at a 1:1 weight ratio (Yang et al., 2016; Li et al., 2020). These three different protein fractions can be separated by centrifugation for proteomics analysis of raw milk (Roncada et al., 2012).

Among the three protein fractions described above, whey accounts for approximately 13∼20% of the total proteins in sheep milk and is generally regarded as having measurable effects on health outcomes such as immunomodulatory, antimicrobial properties, acute inflammatory response, complement activation and innate immune response (Hernández-Ledesma, Ramos & Gómez-Ruiz, 2011; Ha et al., 2015). In addition, the natural digestion of sheep whey proteins in the gastrointestinal tract can generate peptides with a variety of bioactivities, for instance, antihypertensive, opioid, antibacterial, antioxidant, and immunomodulatory activities (Recio & Visser, 2000; Tina et al., 2010). The proteome of cow and sheep whey has attracted extensive research. To date, up to 606 proteins have been identified in sheep whey, while 783 proteins have been found in cow whey (D’Alessandro, Zolla & Scaloni, 2011; Le et al., 2011; Nissen et al., 2013; Reinhardt et al., 2013; Ha et al., 2015). A comparative analysis between the two whey proteomes revealed only 233 common proteins, most of which were associated with immunity (Ha et al., 2015). Gene Ontology (GO) analysis of whey proteins unique to the cow revealed significant involvement in cellular development and metabolism, while proteins unique to sheep were found to function in cellular establishment, signaling, protein maturation, and inflammatory and other immune responses (Ha et al., 2015). Previous proteomic studies on sheep milk have mainly focused on the characterization of the sheep colostrum proteome, MFGMPs, skimmed milk and whey proteins at specific time points (Pisanu et al., 2012; Hernandez-Castellano et al., 2015a) and the proteomic analysis of sheep milk to detect mastitis (Chiaradia et al., 2013). The available differential proteomic studies of milk proteins at different lactation stages have mainly been conducted in bovines (Murgiano et al., 2009; Zhang et al., 2015; Tacoma et al., 2016), while very few such studies have been reported for sheep milk. The changes of protein constituents in the milk whey proteome during the transition from colostrum to mature milk are therefore important for a more detailed understanding of the biological significance of milk whey and can supply a reference for developing lamb weaning strategies and milk replacer formula.

In China, milk production is dominated by cows and dairy goats, while sheep milk is mostly exclusively consumed by newborn lambs and is not used for human consumption. Local sheep breeds in China are usually highly fecund. Hu sheep are excellent representatives of Chinese sheep and are the most widely distributed sheep breed in China. In most cases, ewes of this breed can give birth to 2∼4 lambs per parity and can produce three lambs biennially (Yue, 1996). In addition, Hu sheep can produce 120–150 kg milk in 120–150 days of lactation, which is essential for a high survival rate and fast growth rate of Hu lambs after birth (Yue, 1996). Proteomics have been used widely and effectively to analyze milk-derived proteins, and proteomic technologies have greatly advanced our in-depth knowledge of milk proteins (O’Donnell et al., 2004). In this study, the isobaric tags for relative and absolute quantification (iTRAQ) method was used to explore whey proteome changes in Hu sheep at the six time points of 0, 3, 7, 14, 28 and 56 d after delivery. The method of hierarchical clustering was used to analyze the significantly differentially expressed proteins in the different lactation stages. GO annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to analyze the biological function of differentially expressed proteins (DEPs). The objective of this research was to detect the protein profile of sheep milk during early lactation (within 56 d after delivery) and to provide potential guidance for the early weaning of Hu lambs.

Materials & Methods

Results

Gene ontology analysis of the DEPs

To explore the regulatory mechanism of milk proteins and verify the potential molecular associations among DEPs, we performed GO functional annotation analysis of these proteins according to molecular function, cellular location and biological pathways.

The DEPs were classified according to molecular functions between the milk samples from every two adjacent time points, and the results are shown in Fig. 1. The DEPs identified between 0 d and 3 d presented the most comprehensive molecular functions and were enriched in nine specific functional terms, with the majority being related to binding activity and catalytic activity (accounting for 43% and 30%, respectively), followed by transporter activity, structural molecular activity, antioxidant activity and molecular transducer activity. Other proteins were associated with molecular function regulator, chemoattractant activity and transcription factor activity. The number of molecular function categories of the DEPs in the 3 d vs. 7 d group decreased to 7 that antioxidant activity and transcription factor activity were lost. Furthermore, the function of structural molecular activity was lost in the 7 d vs. 14 d and 14 d vs. 28 d groups but existed in the 28 d vs. 56 d group. Chemoattractant activity only existed in the 0 d vs. 3 d and 3 d vs. 7 d groups.

The GO cellular location classifications of the DEPs identified in Hu sheep at different lactation stages were mainly involved in organelle, cell, extracellular region, membrane-enclosed lumen, synapse, cell junction, macromolecular complex, the extracellular matrix and membrane (Fig. 2). All nine cellular location categories were included in the 0 d vs. 3 d group, and the kinds decreased gradually in other comparison groups. The most prevalent cellular locations were organelle, cell and extracellular region, which together accounted for over 60% in all groups.

There were 17 biological process categories in which the identified DEPs were involved (Fig. 3). The proteins in the 0 d vs. 3 d group exhibited the most complete classifications, and six biological processes exhibited a major change between the remaining comparison groups: behavior, rhythmic process, growth, biological adhesion, multi-organism process and locomotion.

Cluster analysis of the differentially expressed whey proteins

The 121 identified DEPs were analyzed by hierarchical clustering analysis based on their affinity (Fig. 4). The high-abundance proteins α-La and β-Lg exhibited low expression at 0 d, while both of these proteins presented their highest contents at 28 d, which were approximately 2.4 and 4.7 times higher, respectively, than that at 0 d. Expression level changes were also observed for low-abundance proteins between the 6 lactation stages, such as superoxide dismutase [Cu-Zn], airway lactoperoxidase, lipoprotein lipase, and peptidyl-prolyl cis-trans isomerase. In addition, significant differences were found for proteins that were searched according to homology. Lysosomal-associated transmembrane protein 5, G protein-regulated inducer of neurite outgrowth 1 and low-quality proteins such as biorientation of chromosomes in cell division protein 1-like 1 isoform x1 presented a low concentration in 0 d and 3 d milk whey but exhibited high expression in milk at 14 d, 28 d and 56 d after delivery. Proteins such as pyruvate dehydrogenase phosphatase regulatory mitochondrial, connective tissue growth factor, immunoglobulin epsilon-partial, Ig k protein and mortality factor 4 like 1 were almost exclusively present in milk at 0 d after delivery and absent in milk samples at other time points.

Table 6:

Pathway categories of differentially expressed proteins.

Group	Pathway name	KO ID	Protein name
3 d vs. 0 d	Circadian rhythm	oas04710	cryptochrome 1
7 d vs. 3 d	Fatty acid biosynthesis	oas00061	fatty acid synthase (fragment)
	African trypanosomiasis	oas05143	I alpha globin

DOI: 10.7717/peerj.10105/table-6

Discussion

Of those animal milk proteome related researches, most of the studies have focused on cow and goat milk, and few studies have investigated the dynamic changes in sheep milk proteins during lactation stages. The lack of such research has led to the limitation of the sheep protein database (Hernandez-Castellano et al., 2015a). This study analyzed the differential expression of Hu sheep milk whey proteins at different lactation stages. In our research, 121 DEPs were identified, and functional annotation and metabolic pathway annotation were performed to illustrate the molecular functions of the sheep milk whey proteins in infant growth and development. This research enriches the sheep protein database and provides a direction for the exploitation of specific milk protein production.

With the rise of proteomics technology, many studies involving milk protein quantification and qualitative analysis have been conducted, and varieties of research methods have been derived. Researchers identified 149 whey proteins in bovine milk by combinatorial peptide ligand library analysis combined with 1D-SDS-PAGE and 2D-PAGE analyses (D’Amato et al., 2009). Under the ion-exchange approach, 293 unique gene products were identified in bovine colostrum and mature whey (Le et al., 2011). In ovine whey, Scumaci et al. (2015) used a 1D-SDS-PAGE with LC-MS/MS method and identified 343 unique proteins in Appenninic sheep. Ha et al. (2015) used the ProteoMiner Kit to analyze sheep milk whey proteins in depth, and 669 proteins were identified, representing the largest inventory of sheep whey proteins identified to date. Low-abundance proteins are a kind of active substances with low expression that participate in a variety of biological metabolic processes (Righetti, Boschetti & Monsarrat, 2007) but may be covered by high-abundance proteins. ProteoMiner Kit technology can be used to saturate high-abundance proteins quickly and increase the number of minor peptides and proteins that are detectable by LC MS/MS, which is specifically used to analyze low-abundance proteins (Ha et al., 2015). The isobaric tags for relative and absolute quantification (iTRAQ) method is a quantitative proteomics approach with a relatively high throughput that allows simultaneous identification and peptide quantification by measuring the peak intensities of reporter ions via MS/MS (Pottiez et al., 2012). Reinhardt et al. (2013) used iTRAQ technology to quantify protein changes between milk fractions isolated from healthy and S. aureus infected cows, and 748 whey proteins were identified. In this work, we used iTRAQ technology combined with LC-MS/MS and 310 proteins were identified in Hu sheep whey milk at different lactation stages. The number of the identified proteins varies greatly in different researches, which illustrated that milk whey proteins present significant differences between species, and the chosen method also has a great influence on protein detection. The identified proteins were further quantified under the iTRAQ approach in this study, and differentially expressed proteins were found.

The DEPs identified in our research were involved in multiple biological processes, including immunity, growth, disease, lipid metabolism, and protein metabolism. A large number of proteins were indicated to function as immune modulators, such as superoxide dismutase [Cu-Zn], collectin43 isoform x1, haptoglobin isoform x1, fibrinogen alpha/beta chain, complement factor I, complement C3, immunoglobulin, serum amyloid A protein (fragment), and mannose-binding lectin. As the major immune factors, immunoglobulins identified in this research, including immunoglobulin epsilon, immunoglobulin kappa-4 light chain variable region, immunoglobulin lambda-2c light chain variable region, immunoglobulin v lambda chain and immunoglobulin alpha heavy chain, were found to be differentially expressed at different lactation stages, and the expression levels of these proteins were significantly decreased in mature milk. Thus, colostrum is the main source of immune factors for newborns. Roncada et al. (2012) reported that haptoglobins in whey are up-regulated in cows with subclinical and clinical mastitis. Fibrinogen is mainly known as a component of blood that infiltrates into the mammary gland and is increased in plasma after colostrum feeding (Hernandez-Castellano et al., 2015a). The fibrinogen alpha chain is the major precursor of blood clots (Tamzali, Guelfi & Braun, 2001) but also participates in inflammation, stimulating the adhesion, migration, chemotaxis, and phagocytosis of monocytes and macrophages at the point of infection (Akassoglou & Strickland, 2002). Similarly, mannose-binding lectin can alleviate the symptoms of Mycoplasma pneumoniae in sheep, enhance body resistance, and lessen inflammation (Cosenza et al., 2012). Growth-related protein, myosin light chain 6 (fragment), cyclin-g-associated kinase isoform x6, myostatin A, and connective tissue growth factor were also identified as DEPs, and all of these proteins showed decreases after 0 d. Dietary fat intake during infancy is very high, with approximately 50% of the total energy intake being acquired from milk lipids during the first month after birth (Innis, 2007). In the present study, lipoprotein lipase and apolipoprotein E expression levels also presented large differences, and fatty acid synthase (fragment) was identified as a DEP in the 3 d vs. 7 d group. In addition, several proteins involved in protein and nucleotide metabolism, such as histone H2B, are involved in the maintenance of the nucleosome structure of chromosomal fibers in eukaryotes (Maruyama et al., 2006); protein disulfide-isomerase functions in the regulation of protein misfolding; alpha-mannosidase is a key enzyme invovled in the modification of the eukaryotic protein N-glycan, which plays a decisive role in protein synthesis and conformational folding. The decline in the expression levels of these proteins may be correlated with mammary gland activity. In addition, the amounts of DEPs varied greatly in first 7 d after delivery, illustrated that adequate milk intake during this stage is critical to the development of the newborns.

Based on their GO functional annotations, the DEPs were classified in accordance with molecular function, cellular localization and biological pathways. In a previous study, 66% of the DEPs identified in the colostrum and mature milk whey of yak were found to be related to binding activity (Yang et al., 2015). Furthermore, researchers who analyzed ovine colostrum found that approximately 44% of the identified proteins were involved in catalytic activity and that 22.4% of the proteins were involved in binding activity (Scumaci et al., 2015). Our results were highly consistent with previous reports that catalytic activity and binding activity exist throughout the lactation period and account for the largest proportion of the total identified proteins. The molecular function category of the DEPs is most comprehensive in the 0 d vs. 3 d group, indicating that the whey proteins presented the most abundant function at 0 d after delivery. For example, antioxidant activity was only observed in the 0 d vs. 3 d group and was lost in the other groups, which also illustrated that colostrum is very important for providing protection against infections in newborns. Among the characterized processes, the proteins were classified into 17 categories, among which the metabolic process category was the largest and was detected in all time points. Another major process was the response to stimulus, suggesting that these proteins play a complementary role in other immunological and nonimmunological defense mechanisms in the gastrointestinal mucosa (Ogundele, 2001). In a total, the items of biological functions were least in the 56 d vs. 28 d group, which means that the milk whey proteins is at a stable state. Considering the argument of the early weaning time of sheep (Ayadi et al., 2011; Ma et al., 2015), the present study indicated that weaning at 28 d after delivery is feasible.

Conclusions

This study explored the dynamic change tendency of Hu sheep milk whey proteins after delivery for the first time. Using iTRAQ proteomics technology combined with LC-MS/MS methods, a total of 310 whey proteins were identified and 121 DEPs were found. GO annotation analysis revealed that the biological functions of the proteins were most abundant in colostrum (4∼5 h after delivery) and lost in subsequent analysis gradually. The results from this research enriched the proteomics database of sheep milk whey, and are expected to provide a potential guidance for early weaning of lambs.

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