Comparative Site-Specific O-Glycosylation Analysis of the Milk Fat Globule Membrane Proteome in Donkey Colostrum and Mature Milk.

Donkey milk fat globule membrane (MFGM) proteins are a class of membrane-bound secreted proteins with broad-spectrum biofunctional activities; however, their site-specific O-glycosylation landscapes have not been systematically mapped. In this study, an in-depth MFGM O-glycoproteome profile of donkey milk during lactation was constructed based on an intact glycopeptide-centered, label-free glycoproteomics pipeline, with 2137 site-specific O-glycans from 1121 MFGM glycoproteins and 619 site-specific O-glycans from 217 MFGM glycoproteins identified in donkey colostrum and donkey mature milk, respectively. As lactation progressed, the number of site-specific O-glycans from three glycoproteins significantly increased, whereas that of 11 site-specific O-glycans from five glycoproteins significantly decreased. Furthermore, donkey MFGM O-glycoproteins with core-1 and core-2 core structures and Lewis and sialylated branch structures may be involved in regulating apoptosis. The findings of this study reveal the differences in the composition of donkey MFGM O-glycoproteins and their site-specific O-glycosylation modification dynamic change rules during lactation, providing a molecular basis for understanding the complexity and biological functions of donkey MFGM protein O-glycosylation.

Label-free quantitative proteomic analysis of milk fat globule membrane proteins in porcine colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are nutritional components with various biological functions. This study aimed to analyze and compare MFGM proteins in porcine colostrum (PC) and porcine mature milk (PM), via label-free quantitative proteomics. In total, 3917 and 3966 MFGM proteins were identified in PC and PM milk, respectively. A total of 3807 common MFGM proteins were found in both groups, including 303 significant differentially expressed MFGM proteins. Gene Ontology (GO) analysis revealed that the differentially expressed MFGM proteins were mainly related to the cellular process, cell, and binding. The dominant pathway of the differentially expressed MFGM proteins was related to the phagosome according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. These results reveal crucial insights into the functional diversity of MFGM proteins in porcine milk during lactation and provide theoretical guidance for the development of MFGM proteins in the future.

An Intelligent Vascular Disrupting Dendritic Nanodevice Incorporating Copper Sulfide Nanoparticles for Immune Modulation-Mediated Combination Tumor Therapy.

Development of intelligent nanoplatforms that can simultaneously target multiple factors associated with tumor growth and metastasis remains an extreme challenge. Here, an intelligent dendritic nanodevice incorporating both copper sulfide nanoparticles (CuS NPs) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA, a vascular disrupting agent) within the dendrimer internal cavities and surface modified with a targeting agent LyP-1 peptide is reported. The resulting generation 5 (G5) dendrimer-based nanodevice, known as G5-PEG-LyP-1-CuS-DMXAA NPs (GLCD NPs), possess good colloidal stability, pH-sensitive drug release kinetics, and high photothermal conversion efficiency (59.3%). These functional GLCD NPs exert a LyP-1-targeted killing effect on breast tumors by combining CuS-mediated photothermal therapy (PTT) and DMXAA-induced vascular disruption, while also triggering antitumor immune responses through PTT-induced immunogenic cell death and DMXAA-mediated immune regulation via M1 polarization of tumor-associated macrophages and dendritic cell maturation. In addition, with the LyP-1-mediated proapoptotic activity, the GLCD NPs can specifically kill tumor lymphatic endothelial cells. The simultaneous disruption of tumor blood vessels and lymphatic vessels cuts off the two main pathways of tumor metastasis, which plays a two-pronged role in inhibiting lung metastasis of the breast cancer model. Thus, the developed GLCD NPs represent an advanced intelligent nanoformulation for immune modulation-mediated combination tumor therapy with potential for clinical translations.

Comparative effectiveness of different probiotics supplements for triple helicobacter pylori eradication: a network meta-analysis.

Background: Probiotics has been reported as an effective supplement for Helicobacter pylori eradication. However, knowledge of their comparative efficacy is still lacking. Aim: In this study, we used network meta-analysis of current probiotics supplement used in standard triple therapy to assess and rank their comparative effectiveness. Methods: All randomized controlled trials from three main databases (PubMed, Embase and Cochrane Library) up to April 2022 were collected and filtered to meet our criterion. We used Bayesian network meta-analysis to evaluate the eligible randomized controlled trials and gave a rank for the efficiency and incidence of side effects of each probiotics supplement. The ranking probability for each therapy was assessed by means of surfaces under cumulative ranking values. Subgroup analysis was conducted to evaluate other possible influencing factors. Results: 34 eligible randomized controlled trials entered the following meta-analysis, including 9,004 patients randomized to 10 kinds of therapies. Result showed that most probiotics added therapies had better outcomes than triple therapy, among which Bifidobacterium-Lactobacillus and Bifidobacterium-Lactobacillus-Saccharomyces adjuvant therapy could obtain comprehensive benefit with high eradication rate (78.3% and 88.2% respectively), and cause few side effects. Combination of different probiotics, adding probiotics before or after triple therapy and longer duration of probiotics can improve therapeutic effect in H.pylori infected individuals. Conclusion: For triple therapy of H.pylori infection, adding probiotics can increase eradication rate and bring protective effect. Considering the overall influence, Bifidobacterium-Lactobacillus or Bifidobacterium-Lactobacillus-Saccharomyces therapy can be a better choice in improving H.pylori eradication process.

Characterization and comparison site-specific N-glycosylation profiling of milk fat globule membrane proteome in donkey and human colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are highly glycosylated and involved in various biological processes within the body. However, information on site-specific N-glycosylation of MFGM glycoproteins in donkey and human milk remains limited. This study aimed to map the most comprehensive site-specific N-glycosylation fingerprinting of donkey and human MFGM glycoproteins using a site-specific glycoproteomics strategy. We identified 1,360, 457, 2,617, and 986 site-specific N-glycans from 296, 77, 214, and 196 N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM), respectively. Bioinformatics was used to describe the structure-activity relationships of DC, DM, HC, and HM MFGM N-glycoproteins. The results revealed differences in the molecular composition of donkey and human MFGM N-glycoproteins and the dynamic changes to site-specific N-glycosylation of donkey and human MFGM glycoproteins during lactation, deepening our understanding of the composition of donkey and human MFGM N-glycoproteins and their potential physiological roles.

Quantitative label-free site-specific glycoproteomic analysis of the milk fat globule membrane protein in human colostrum and mature milk.

Human milk fat globule membrane (MFGM) proteins, which are N-glycosylated, play essential roles in neonatal development and physiological health. However, the profiles and landscape changes in the site-specific N-glycosylation of human MFGM proteins during lactation remain unclear. Therefore, in this study, based on an intact glycopeptide-centred strategy, 2617 unique site-specific N-glycans of 221 MFGM glycoproteins in human colostrum and 986 unique site-specific N-glycans of 200 MFGM glycoproteins in mature milk were characterised and quantified using label-free glycoproteomics. With milk maturation, 33 site-specific N-glycans on 10 N-glycoproteins increased significantly, and 113 site-specific N-glycans on 25 N-glycoproteins decreased significantly. Moreover, human MFGM glycoproteins with core-α1,6-fucosylated structures and Lewis and sialylated branching structures play a role in the biological processes of antigen processing and presentation. This study reveals the dynamic changes in human MFGM protein N-glycosylation patterns during lactation. Meanwhile, the study deepens our understanding of site-specific N-glycosylation of human MFGM glycoproteins. The results of the study provide a background reference for the development of infant formulas.

Quantitative phosphoproteome analysis reveals differential whey phosphoproteins of bovine milk during lactation.

Whey proteins in bovine milk, as the most widely used nutritional components for infant formulae, have been paid more attention. However, the phosphorylation of proteins in bovine whey during lactation has not been thoroughly researched. In this study, a total of 185 phosphorylation sites on 72 phosphoproteins were identified in bovine whey during lactation. 45 differentially expressed whey phosphoproteins (DEWPPs) in colostrum and mature milk were focused on by bioinformatics approaches. Gene Ontology annotation indicated that blood coagulation, extractive space, and protein binding played a key role in bovine milk. The critical pathway of DEWPPs was related to the immune system according to KEGG analysis. Our study investigated the biological functions of whey proteins from a phosphorylation perspective for the first time. The results elucidate and increase our knowledge of differentially phosphorylation sites and phosphoproteins in bovine whey during lactation. Additionally, the data might offer fresh insight into the development of whey protein nutrition.

A new sight to explore site-specific N-glycosylation in donkey colostrum milk fat globule membrane proteins with glycoproteomics analysis.

Donkey colostrum milk fat globule membrane (DCMFGM) proteins are involved in multiple biological functions. However, the effect of N-glycosylation on their physiological properties are unknown. The aim of this study was to map the DCMFGM protein site-specific N-glycosylation landscape using a label-free glycoproteomic approach. A total of 1,443 unique intact N-glycopeptides mapping to 453 unique N-glycosites on 336 N-glycoproteins were identified. The macro- and microheterogeneity of DCMFGM glycoproteins were explored at the N-glycosite level and the site-specific N-glycan level, respectively, and it was found that the N-glycosylation profiles of the DCMFGM proteins varied based on subcellular localisation and protein domain types. Our findings reveal the heterogeneity and functional diversity of N-glycosylation of DCMFGM proteins and provide theoretical support for the promotion of DCMFGM proteins as a functional food ingredient.

Polydopamine-Coated Laponite Nanoplatforms for Photoacoustic Imaging-Guided Chemo-Phototherapy of Breast Cancer.

Theranostic nanoplatforms combining photosensitizers and anticancer drugs have aroused wide interest due to the real-time photoacoustic (PA) imaging capability and improved therapeutic efficacy by the synergistic effect of chemotherapy and phototherapy. In this study, polydopamine (PDA) coated laponite (LAP) nanoplatforms were synthesized to efficiently load indocyanine green (ICG) and doxorubicin (DOX), and modified with polyethylene glycol-arginine-glycine-aspartic acid (PEG-RGD) for PA imaging-guided chemo-phototherapy of cancer cells overexpressing αvß3 integrin. The formed ICG/LAP-PDA-PEG-RGD/DOX nanoplatforms showed significantly higher photothermal conversion efficiency than ICG solution and excellent PA imaging capability, and could release DOX in a pH-sensitive and NIR laser-triggered way, which is highly desirable feature in precision chemotherapy. In addition, the ICG/LAP-PDA-PEG-RGD/DOX nanoplatforms could be uptake by cancer cells overexpressing αvß3 integrin with high specificity, and thus serve as a targeted contrast agent for in vivo PA imaging of cancer. In vivo experiments with 4T1 tumor-bearing mouse model demonstrated that ICG/LAP-PDA-PEG-RGD/DOX nanoplatforms exhibited much stronger therapeutic effect and higher survival rate than monotherapy due to the synergetic chemo-phototherapy under NIR laser irradiation. Therefore, the reported ICG/LAP-PDA-PEG-RGD/DOX represents a promising theranostic nanoplatform for high effectiveness PA imaging-guided chemo-phototherapy of cancer cells overexpressing αvß3 integrin.

Characterization and comparison of lipids in bovine colostrum and mature milk based on UHPLC-QTOF-MS lipidomics.

Lipids in bovine milk have several biological activities, with implications for human health and the physical functionality of foods. However, alterations in the lipid profile of bovine milk during lactation are not well-studied. This study aimed to identify differences in lipids between bovine colostrum and mature milk, using a lipidomics approach. Using an advanced mass spectrometry-based quantitative lipidomics approach, 335 lipids assigned to 13 subclasses were characterized in bovine colostrum (BC) and mature milk (BM). In total, 63 significantly differential lipids (SDLs) were identified. Among the 63 SDLs, the levels of 21 lipids were significantly lower in BM than in BC, including 5 glycerophosphatidylethanolamines (PEs), 1 glycerophosphatidylglycerol (PG), and 15 triacylglycerols (TGs). The levels of the remaining 42 lipids increased in BM, including 1 cardiolipin (CL), 9 diacylglycerols (DGs), 9 dihexosylceramides (Hex2Cers), 3 hexosylceramides (HexCers), 3 glycerophosphatidic acids (PAs), 2 glycerophosphatidylcholines (PCs), 12 PEs, and 3 TGs. Furthermore, the correlations and related metabolic pathways of these 63 SDLs were analyzed to explore the mechanisms that alter bovine milk lipids during lactation. The seven most relevant pathways identified herein, ranked in accordance with their degree of influence on lactation, were glycerophospholipid metabolism, sphingolipid metabolism, glycerolipid metabolism, glycosylphosphatidylinositol-anchor biosynthesis, linoleic acid metabolism, alpha-linolenic acid metabolism, and arachidonic acid metabolism. Our results provide essential insights into mechanisms underlying alterations in bovine milk lipids during different lactation periods, along with practical information of specific nutrition and quality assessments for the dairy industry.

Donkey milk inhibits triple-negative breast tumor progression and is associated with increased cleaved-caspase-3 expression.

Donkey milk is considered an ideal substitute for human milk and is considered a potential complementary dairy product for the treatment of a variety of human diseases, including cancer. The purpose of this study was to investigate the inhibitory effect of donkey colostrum (DC) and mature milk (DM) on 4T1 triple-negative breast cancer (TNBC) tumors in mice. Metabolomics analyses showed that a total of 476 possible metabolites were found in both types of milk. Among them, 34 differential metabolites were identified, including 25 up-regulated and 9 down-regulated metabolites in the DC compared with DM. Both DC and DM are rich in many known anticancer constituents. The inhibitory effects of DC and DM on 4T1 primary tumors and the relative organ weight of the liver and lungs were determined by measuring the volume of primary tumors and weighing the liver and lungs. Both DC and DM significantly reduced both the primary tumor size and relative organ weight of the liver and lungs in 4T1 mice without affecting the bodyweight of mice. When the expression of cleaved caspase-3, Bax, and MMP2 was investigated by immunohistochemistry, the results showed that DC and DM inhibited the progression of 4T1 tumors by inducing the expression of cleaved-caspase-3 and Bax, and inhibiting the expression of MMP2 and CD31. Our data suggest that DC and DM inhibit the growth and metastasis of mouse 4T1 tumors by inducing apoptosis.

Quantitative Phosphoproteomics of Milk Fat Globule Membrane in Human Colostrum and Mature Milk: New Insights into Changes in Protein Phosphorylation during Lactation.

Phosphorylation is a widespread posttranslational protein modification and is important in various biological processes. However, milk fat globule membrane (MFGM) phosphoproteins have not been explored systematically in human milk. Here, we used quantitative phosphoproteomics to analyze phosphorylation sites in human MFGM proteins and their differences at different stages of lactation; 305 phosphorylation sites on 170 proteins and 269 phosphorylation sites on 170 proteins were identified in colostrum and mature MFGM, respectively. Among these, 71 phosphorylation sites on 48 proteins were differentially expressed between the different stages of lactation. Osteopontin in human MFGM was the most heavily phosphorylated protein, with a total of 39 identified phosphorylation sites. Our results shed light on phosphorylation sites, composition, and biological functions of MFGM phosphoproteins in human colostrum and mature milk, and provide novel insights into the crucial roles of protein phosphorylation during infant development.

Comparative analysis of whey proteins in donkey colostrum and mature milk using quantitative proteomics.

Donkey milk is attracting increasing attention as a nutritional milk source similar to human milk. In this study, we carried out qualitative and quantitative analysis of the donkey whey proteome using a label-free proteomic approach, combined with parallel reaction monitoring (PRM) as a validation method. A total of 300 whey proteins were identified in donkey colostrum (DC) and donkey mature (DM) milk, of which 18 were differentially expressed (P < 0.05) between the two types of milk. Gene ontology (GO) analysis showed that differentially and uniquely expressed proteins were mainly involved in cellular processes, response to stimulus, metabolic processes, and biological regulation. Their molecular functions included binding, catalytic activity, and molecular functional regulation, and their main annotated areas of origin were the cell, cell-part, and the extracellular region. Most differentially and uniquely expressed proteins were linked with malaria, systemic lupus erythematosus, or antigen processing and presentation. Our results provide insight into the complexity of the donkey whey proteome and molecular evidence for nutritional differences between different lactation stages.

Quantitative proteomic analysis of milk fat globule membrane (MFGM) proteins from donkey colostrum and mature milk.

The composition and functions of milk fat globule membrane (MFGM) proteins are important indicators of the nutritional quality of milk. However, these characteristics of MFGM proteins in donkey milk are unknown at different lactation periods. We characterized and identified MFGM proteins in donkey milk at two lactation periods using label-free proteomics. A total of 947 MFGM proteins were found. There were 902 and 913 MFGM proteins in donkey colostrum and mature milk, respectively. The differentially expressed MFGM proteins were classified into different Gene Ontology annotations. The biological process subgroups containing the most MFGM proteins mainly included cellular process, metabolic process, biological regulation, and regulation of biological processes. Donkey MFGM proteins participated in several Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways at different lactation stages, such as endocytosis, thermogenesis, Alzheimer's disease, cancer, and human papillomavirus infection. The knowledge gained in this study may provide theoretical insights and guidance for the future development of novel infant formulae.

Characterization and comparison of milk fat globule membrane N-glycoproteomes from human and bovine colostrum and mature milk.

Human and bovine milk fat globule membrane (MFGM) proteins have been identified and characterized; however, their glycosylation during lactation remains unclear. We adopted a glycoproteomics approach to profile and compare MFGM N-glycoproteomes in human and bovine milk during lactation. A total of 843, 718, 614, and 273 N-glycosite peptides corresponding to 465, 423, 334, and 176 glycoproteins were identified in human colostrum, human mature milk, bovine colostrum, and bovine mature milk, respectively. The biological functions of these MFGM N-glycoproteins were revealed through bioinformatics. Substantial differences were observed between human and bovine milk, and immune-related MFGM N-glycoproteins varied between colostrum and mature milk from both species. Our results expand current knowledge of MFGM N-glycoproteomes, and further demonstrate the complexity and biological functions of MFGM N-glycosylation. These data can provide references for the application of bovine MFGM N-glycoproteins in infant formula to resemble human milk and in functional foods.

Characterization and comparison of whey N-glycoproteomes from human and bovine colostrum and mature milk.

Milk glycoproteins are crucial nutrients with a variety of functions. However, whey N-glycoproteomes in human and bovine milks have not been characterized during lactation. Herein, using lectin enrichment and liquid chromatography tandem mass spectrometry, 68, 58, 100, and 98 N-glycoproteins were identified in human colostrum and mature milk as well as bovine colostrum and mature milk whey. Gene Ontology and KEGG pathway analyses were used to elucidate the biological functions of whey N-glycoproteins in human and bovine colostrum and mature milks. Whey N-glycoproteomes differed dramatically between human and bovine milks and across lactation stages. The conserved and specific whey N-glycoproteins in all four sample types were also determined. Our results improve understanding of the properties and biological functions of whey N-glycoproteins in human and bovine milk and colostra, and provide insight into the potential application of some N-glycoproteins in infant formulae at different stages of development.

Quantitative analysis of amino acids in human and bovine colostrum milk samples through iTRAQ labeling.

BACKGROUND: The types and quantity of proteins vary widely between bovine and human milk, with corresponding differences in free and hydrolytic amino acids. In this study, the free and hydrolytic amino acids of bovine and human colostrum were for the first time qualitatively and quantitatively determined using isobaric tags for relative and absolute quantification technology combined with liquid chromatography tandem mass spectrometry detection. RESULTS: Total free amino acid content was 0.32 g L-1 and 0.63 g L-1 in bovine and human colostrum respectively, with free amino acid content in human colostrum twice that of bovine colostrum. However, total hydrolytic amino acid content was 4.2 g L-1 and 2.2 g L-1 in bovine and human colostrum respectively. We found that the hydrolytic amino acid content in bovine colostrum was higher than that in human colostrum; however, the amount of free amino acids and the overall amino acid content in human colostrum were respectively substantially higher and more varied than in bovine colostrum. CONCLUSION: Our findings revealed differences between bovine and human colostrum, with these data providing the basis for further research into amino acid metabolomics and infant formula. © 2018 Society of Chemical Industry.

Quantitative N-glycoproteomics of milk fat globule membrane in human colostrum and mature milk reveals changes in protein glycosylation during lactation.

Milk fat globule membrane (MFGM) proteins have recently gained increasing attention, due to their significant biological function. However, the glycosylation of proteins in human MFGM during lactation has not been studied in detail. In this study, through mass spectroscopy-based N-glycoproteomics, we analyzed protein glycosylation of human MFGM. A total of 912 N-glycosylation sites on 506 N-glycoproteins were identified in human colostrum and mature milk MFGM. Among them, 220 N-glycoproteins with 304 N-glycosylation sites were differentially expressed in colostrum and mature milk MFGM. Gene Ontology (GO) analysis revealed various biological processes, cellular components, and molecular functions of the differentially expressed N-glycoproteins. Specifically, these glycoproteins were involved in biological processes such as single-organism processes, biological regulation, regulation of biological processes, response to stimulus and localization; were cellular components in organelles, membranes, and the extracellular region; and had different molecular functions such as protein binding, receptor activity, and hydrolase activity. KEGG pathway analysis suggested that the majority of the differentially expressed N-glycoproteins were associated with phagosome, cell adhesion molecule and some disease-related pathways. Our results provide an in-depth understanding of the quantitative changes in N-glycosylation of proteins in human colostrum and mature MFGM, and extend our knowledge of the N-glycoproteome and of the distribution of N-glycosylation sites in human MFGM during lactation, providing insight into the biological functions of the highlighted glycoproteins.

Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

Comparative proteomic analysis of milk-derived exosomes in human and bovine colostrum and mature milk samples by iTRAQ-coupled LC-MS/MS.

Exosomes are membranous vesicles found in biological fluids with important functions. However, milk-derived exosome proteins from humans and bovines have not been studied in detail. The advanced iTRAQ proteomic approach was used to analyze milk-derived exosomes in human and bovine colostrum and mature milk samples. A total of 920 milk exosome proteins were identified and quantified. Among these, 575 differentially expressed exosome proteins (P<0.05) were found. Multivariate analysis, gene ontology (GO) annotation and the KEGG pathway were used to interpret the identified proteins. The major biological processes involved were: response to stimulus (22%), localization (16%), establishment of localization (14%), and cellular component organization (14%). Cellular components engaged in intracellular (31%) and intracellular part (31%). The most prevalent molecular function mainly touched upon binding (52%). Milk exosome proteins participated in several KEGG pathways containing ribosome, regulation of actin cytoskeleton, glycolysis/gluconeogenesis, leukocyte transendothelial migration, aminoacyl-tRNA biosynthesis, pentose phosphate pathway, galactose metabolism and fatty acid biosynthesis. These results provide important information on human and bovine milk exosomes, and increase knowledge on the proteomes of these exosomes across different lactation stages, which could provide potential directions for newborn milk powder, biological markers and functional foods.