In this study, we applied a label-free mass spectrometry-based quantitative proteomics approach to compare the whey proteome profiling of DM with HM or CM. In total, 249 whey proteins were found to be significantly different between DM and HM; 418 whey proteins were significantly different between DM and CM. These DEPs were involved in lipid metabolic process, regulation of cytokine production, chemical homeostasis and catabolic process. These results may provide valuable information in the composition of milk whey proteins in DM, HM and CM, especially for low abundant components, and expand our knowledge of different biological functions between DM and HM or CM.
DM was characterized by a particularly high whey protein content which was rich in lysozyme, α-lactalbumin (α-La), β-lactoglobulin (β-Lg) and serum albumin (SA) [1]. In our data, we also found these whey proteins in DM were significantly higher than HM and CM. Among them, the amount of lysozyme in DM has proved to be higher with respect to that in CM, DM and goat’s milk [12]. As a powerful antibacterial protein, lysozyme plays an important role in the intestinal immune response. DM lysozyme belongs to C-type calcium-binding lysozyme and could bind calcium ions which leads to more stable complex with an enhanced antimicrobial activity [13]. β-Lg is the major whey protein in DM and CM, whereas in HM the β-Lg is absent [14]. However, in present study β-Lg was identified in HM with low abundance. The reason for this probably is that β-Lg can be detected in HM after dairy products ingestion [15, 16].
According to the GO and KEGG pathway enrichment analysis, DEPs in DM vs HM group and DM vs CM group were all mainly involved lipid binding, lipid metabolic process and cholesterol pathway. Among these DEPs, a family of apolipoproteins (Apos), namely, apolipoprotein A1 (ApoA1), ApoA2, ApoA4, ApoE and ApoH, were significantly upregulated in DM. ApoA1 and ApoA2 are the major proteins in high-density lipoproteins (HDL) [17]. ApoA1 has multiple beneficial functions, including potent antioxidant, anti-inflammatory, antiviral and antibacterial activities in blood [18] [19, 20]. Recently, ApoA1 was found in HM and DM. Kim et al. identified that ApoA1 interacts with cholesterol in HM, provides antioxidant activity and improves embryo survivability [21]. In DM milk fat globule membrane (MFGM), ApoA1 was upregulated in colostrum compared with mature milk [22]. ApoE and ApoH are also crucial elements in the lipoprotein metabolism and cholesterol transport [23, 24]. The higher concentrations of ApoE in CM and HM were found in early lactation, and significantly decreased over lactation, indicating the importance of cholesterol in the development of the neonate [25, 26]. Cholesterol plays an important role in the synthesis of vitamin D and the steroid hormones, which is critical to the development of the newborns [26]. In our study, DM provides a higher level of cholesterol transporters than HM and CM, which may help the newborns acquire a large amount of cholesterol. In addition to cholesterol transport function, recent work in both cell culture and in mice indicates that HDL (mainly ApoA1, and ApoA2) have anti-atherogenic effects and cause robust activation of endothelial nitric oxide (NO) synthase [17]. ApoE deficiency in mice results in a profound susceptibility to atherosclerosis [27]. Meanwhile, DM could induce human peripheral blood mononuclear cells (PBMCs) to release NO, which is very useful in the prevention of atherosclerosis [3]. Therefore, we hypothesized that these upregulated Apos might contribute the effect to atherosclerosis prevention induced by DM.
Milk provides large amounts of bioactive components to the infants in the critical phase of immunological immaturity of the newborn, particularly for the immune system of mucous membranes. Breastfeeding protects infants against infections mainly via secretory IgA (SIgA) antibodies. IgM antibodies, the second most abundant immunoglobulin in human colostrum, are also important in protecting the mucosal surfaces of infants through its reaction with viruses and bacteria [28]. Both dimeric IgA and pentameric IgM are transported across the epithelial cells into the milk by the polymeric Ig receptor (pIgR), expressed on the basolateral surface of mammary epithelial cells [29, 30]. In addition to the heavy and light chains, dimeric IgA and pentameric IgM contain a small polypeptide known as the joining (J) chain, which plays an important role in the generation of secretory antibodies because it provides them with the capacity to bind the pIgR [31]. This peptide can be produced by immunocytes of all Ig isotypes, but it becomes incorporated only into IgA and pentameric IgM [32]. Moreover, J chain expression may be a marker for B-cell clones derived from mucosa-associated lymphoid tissue, as there is a positive correlation between the production of polymeric IgA, IgG or IgD-producing cells and J chain [32, 33]. In our data, J chain were significantly upregulated in DM vs HM group (18.0-fold) and DM vs CM group (3.3-fold) respectively. We speculated that this high level of J chain production might reflects the abundant IgA, IgM or other immunoglobulins in DM and contributes to explain the previous studies indicating DM intake improves anti-inflammatory defenses in rats [34]. Other abundant whey proteins related to immune responses in DM included secreted phosphoprotein 1 (SPP1), complement C2 and complement C3. SPP1, present in significant amounts in breast milk [35], is a multifunctional protein involved in cell-mediated immune responses and anti-inflammatory responses [36, 37]. Complement proteins are helpful for establishment of a natural immune system in newborns [38]. The presence of abundant immunological factors in DM are helpful for the newborns to establish an immune system against microbial infection to adapt to the new environment to prevent diseases.