Milk Fat Globule Membrane Proteome Research Articles

Changes in milk fat globule membrane proteins along lactation stage of Laoshan dairy goat

The milk fat globule membrane (MFGM) is a complex structure with numerous functions, and its composition is affected by many factors. There have been few systematic investigations on goat MFGM proteome profiling during lactation. Individual milk samples from 15 healthy dairy goats were obtained at six lactation time points for investigation of the MFGM proteome using both data-independent acquisition (DIA) and data-dependent acquisition (DDA) proteomics techniques combined with multivariate statistical analysis. Using the DIA method, 890 variably abundant MFGM proteins were discovered throughout the lactation cycle. From 1 to 240 d, butyrophilin subfamily 1 member A1, lipoprotein lipase, perilipin-2, and adipose triglyceride lipase were upregulated, while APOE, complement C3, clusterin, and IgG were downregulated. Furthermore, from 1 to 90 d, annexin A1, annexin A2, and antithrombin-III were downregulated, then upregulated by d 240. Albumin had a high degree of connectedness, indicating that it was a key protein, according to protein–protein interaction research. Overall, our findings gave new insights into the biological features of MFGM protein in goat milk throughout lactation, which may aid in the creation of specialized MFGM products and infant formula.

Effects of maternal and perinatal factors on human milk fat globule membrane proteome: A data independent acquisition approach

Milk fat globule membrane proteins (MFGMP) are essential for supporting infants’ growth and development. The composition of MFGMP may vary depending on maternal and perinatal factors. However, there is a lack of research on the effects of parity, maternal age, and mode of delivery on MFGMP. This study attempts to address this knowledge gap by utilizing a data-independent acquisition (DIA) approach to perform proteomics comparisons of human MFGMP. Mature human milk (MHM) was collected from Chinese women with different parities (multiparous vs. primiparous, MP vs. PP), maternal reproductive ages (age ≥30 vs. age <30, MA1 vs. MA2), and delivery modes (cesarean delivery vs. vaginal delivery, CD vs. VD). We found variations in MFGMP between different cohorts. Differentially expressed proteins (DEP) in MHM of primiparous and multiparous women were mostly related to innate immune response which provides the first line of defense against pathogens. Higher abundances of these DEP were found in the MHM of multiparous women. Meanwhile, DEP in MHM of women with different delivery modes were primarily related to inflammatory response and innate immune response. We postulate that upregulation of the defense response-related DEP in MHM of women who had cesarean delivery was in relation to modulation of the cesarean delivery-induced inflammatory response and to strengthen the immune defense of newborns.

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.

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.

Milk Fat Globule Membrane Proteome and Micronutrients in the Milk Lipid Fraction: Insights into Milk Bioactive Compounds

Milk lipids are composed of milk fat globules (MFGs) surrounded by the milk fat globule membrane (MFGM). MFGM protects MFGs from coalescence and enzymatic degradation. The milk lipid fraction is a “natural solvent” for macronutrients such as phospholipids, proteins and cholesterol, and micronutrients such as minerals and vitamins. The research focused largely on the polar lipids of MFGM, given their wide bioactive properties. In this review we discussed (i) the composition of MFGM proteome and its variations among species and phases of lactation and (ii) the micronutrient content of human and cow’s milk lipid fraction. The major MFGM proteins are shared among species, but the molecular function and protein expression of MFGM proteins vary among species and phases of lactation. The main minerals in the milk lipid fraction are iron, zinc, copper and calcium, whereas the major vitamins are vitamin A, β-carotene, riboflavin and α-tocopherol. The update and the combination of this knowledge could lead to the exploitation of the MFGM proteome and the milk lipid fraction at nutritional, biological or technological levels. An example is the design of innovative and value-added products, such as MFGM-supplemented infant formulas.

Proteome of milk fat globule membrane and mammary gland tissue in goat fed different lipid supplementation

This study was conducted to assess the effect of lipid supplementation on the proteomic profile of milk fat globule membrane (MFGM) and mammary gland tissue (MGT) in lactating goats. Thirty-eight crossbred goats received a total mixed ration (TMR) based on chopped elephant grass and concentrate during 35 days post-partum. They were divided into three diet groups two weeks after kidding, namely, Control group (CG; n = 12), fed baseline TMR; Flaxseed group (FG; n = 13), fed ground flaxseed added to the TMR for 21 days (12 % total dry matter); and microalgae Chlorella group (MCG; n = 13), fed microalgae Chlorella added to the TMR for 21 days (0.05 % live weight). Blood plasma concentrations of total protein, albumin, and glutathione peroxidase were determined. Weekly milk samples were used to estimate milk yield and protein and fat levels. MGT samples were biopsied on the 35th day of the experiment. The protein profile of MFGM and MGT was assessed using 1D SDS–PAGE and mass spectrometry. The MCG group showed lower dry matter intake and higher milk fat than the other diets. The milk protein level was not affected by the diet group. The 22 protein bands identified in MFGM showed no difference between diets. The multivariate model selected eight protein bands (VIP > 1) to segregate the diet groups. Nine out of 44 protein bands identified in MGT showed lower expression in the FG group, which had a lipid level of the diet of 8 %. The multivariate model involved 11 band proteins (VIP > 1) in the diet group segregation. Thus, the increased lipid level in the diet or milk of lactating goats had no marked effects on the MFGM proteome. High lipid diet interfered with the expression of proteins in the mammary glands, which points to the greater sensitivity of this organ to the effort of lipotoxicity.

Differences in milk fat globule membrane proteins among Murrah, Nili-Ravi and Mediterranean buffaloes revealed by a TMT proteomic approach

Milk fat globule membrane (MFGM) proteins contribute to human nutrition and health. However, differences in MFGM proteome among the buffalo breeds are unknown. In this study, MFGM proteins from Murrah, Nili-Ravi and Mediterranean buffaloes were identified using a tandem mass tag proteomic approach to characterize the components and their potential activities. A total of 1258 MFGM proteins were identified and 103 differentially expressed proteins were found in the studied buffalo breeds. Of these, toll-like receptor 2 had higher abundance in Mediterranean milk; endoplasmic reticulum resident protein 29 had higher levels in Nili-Ravi milk; uromodulin had higher abundance in Murrah milk. A number of 781 newly proteins in buffalo milk exerted antioxidant, antibacterial, and anti-inflammatory activities. Our findings reveal the component complexity and functional diversity of MFGM proteins in different buffalo breeds. Thus, our data may path the way to promote raw buffalo milk as a healthy and functional dairy product.

Comparative Proteomics of Milk Fat Globule Membrane (MFGM) Proteome across Species and Lactation Stages and the Potentials of MFGM Fractions in Infant Formula Preparation.

Milk is a lipid-in-water emulsion with a primary role in the nutrition of newborns. Milk fat globules (MFGs) are a mixture of proteins and lipids with nutraceutical properties related to the milk fat globule membrane (MFGM), which protects them, thus preventing their coalescence. Human and bovine MFGM proteomes have been extensively characterized in terms of their formation, maturation, and composition. Here, we review the most recent comparative proteomic analyses of MFGM proteome, above all from humans and bovines, but also from other species. The major MFGM proteins are found in all the MFGM proteomes of the different species, although there are variations in protein expression levels and molecular functions across species and lactation stages. Given the similarities between the human and bovine MFGM and the bioactive properties of MFGM components, several attempts have been made to supplement infant formulas (IFs), mainly with polar lipid fractions of bovine MFGM and to a lesser extent with protein fractions. The aim is thus to narrow the gap between human breast milk and cow-based IFs. Despite the few attempts made to date, supplementation with MFGM proteins seems promising as MFGM lipid supplementation. A deeper understanding of MFGM proteomes should lead to better results.

Shifts in the Holstein dairy cow milk fat globule membrane proteome that occur during the first week of lactation are affected by parity

BackgroundThe milk fat globule membrane (MFGM) proteomes of colostrum and transition milk are rich sources of proteins that are likely important for neonatal calf health. In addition, characterization of these proteomes could also yield valuable information regarding mammary gland physiology of the early postpartum lactating cow. The objectives of this research were to characterize the MFGM proteomes of colostrum and transition milk through sample collections at four timepoints postpartum, including the first milking (M1, colostrum), second milking (M2, transition milk), fourth milking (M4, transition milk), and fourteenth milking (M14, mature milk), and compare these proteomes between multiparous (MP; n = 10) and primiparous (PP; n = 10) Holstein dairy cows. Isolated MFGM proteins were labeled using Tandem Mass tagging and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein identification was completed using MASCOT and Sequest in Proteome Discoverer 2.2. The scaled abundance values were analyzed using PROC MIXED in SAS to determine the effects of milking (MIL), parity (PAR), and MIL × PAR. The adaptive false-discovery rate (FDR)-adjusted P values were determined using PROC MULTTEST. Protein characterization and bioinformatic analysis were completed using a combination of PANTHER, Blast, and Uniprot.ResultsA total of 104 common proteins were identified in each of the MFGM samples. Statistical analysis revealed that 70.2% of identified proteins were affected by MIL. Of these, 78.1% were lower in M14 compared with M1, including immune-related proteins lactotransferrin, lactadherin and hemopexin. Parity affected 44.2% of proteins. Of the proteins affected by PAR, 84.8% were higher in MP cows compared with PP cows, including apolipoprotein E and histones 2A, 2B, 3, and 4 b. Butyrophilin subfamily 1 member 1A and annexin 5 were higher in samples from PP cows. Milking × parity affected 32.7% of identified proteins, including lactotransferrin, gelsolin, vitamin D binding protein, and S100 proteins.ConclusionsThis research supports previous findings that the Holstein MFGM proteome changes rapidly during the first week of lactation. In addition, this research identifies the impact of parity on the colostrum and transition milk MFGM proteomes, which may be important for milk-fed calf health or for the identification of protein biomarkers for mammary functionality.

Comparative analysis of the skim milk and milk fat globule membrane proteomes produced by Jersey cows grazing pastures with different plant species diversity

The objective of this experiment was to identify and characterize the bovine milk proteome within the skim milk fraction and milk fat globule membrane (MFGM)-associated fraction from 16 organically certified lactating Jersey cows after a short term of grazing pastures with or without annual forage crops (AFC). Cows were offered a partial mixed ration (~60% of dry matter intake) and approximately 40% of their total dry matter intake as herbage. Eight cows were offered a cool-season grass–legume herbage (GLH), which included orchardgrass (Dactylis glomerata), timothy (Phleum pratense), Kentucky bluegrass (Poa pratensis), and white clover (Trifolium repens). The other 8 cows were offered the same GLH strip-tilled with the AFC, including oat (Avena sativa), millet (Pennisetum glaucum), teff (Eragrostis tef), buckwheat (Fagopyrum esculentum), and chickling vetch (Lathyrus sativus). Milk samples were collected from each cow during a.m. and p.m. milkings on d 19 to 21 of grazing, and composite milk samples per cow were analyzed for (1) the high-abundance milk protein profile, (2) the skim milk low-abundance protein-enriched proteome, and (3) the MFGM proteome. Of the 443 proteins identified in the skim and MFGM proteomes, 433 were included in statistical analysis, including 68 proteins identified in the skim milk fraction and 365 in the MFGM-associated fraction. Analysis of the skim and MFGM proteomes encompassed unique gene ontology profiles and proportions of functional classifications. In response to diet, αS1-casein as well as 8 low-abundance proteins were present in higher concentration or abundance in milk from cows grazing the GLH strip-tilled with the AFC compared with milk from cows grazing GLH, suggesting that even short-term grazing of pastures including some AFC may affect the milk proteome.

Characterization of the milk fat globule membrane proteome in colostrum and mature milk of Xinong Saanen goats

Xinong Saanen goat milk is a major source of milk in the Chinese dairy industry. Milk fat globule membrane (MFGM) proteomes of goat colostrum and mature milk were analyzed and compared using proteomic technology. A total of 543 and 585 proteins were identified in goat colostrum and mature milk, respectively. Functional category analyses revealed that most of the MFGM proteins in both colostrum and mature milk were related to phosphoprotein and acetylation. The biological process of translation, cellular component of extracellular exosome, and molecular function of poly(A) RNA binding were the main gene ontology annotations of both colostrum and mature milk. Pathways associated with disease and genetic information processing involved large number of proteins in colostrum and mature milk, and more metabolism-related pathways were observed in mature milk. Protein-protein interaction network analyses showed that ribosome was abundant in both colostrum and mature milk. Colostrum showed more functions associated with protein processing in the endoplasmic reticulum, whereas mature milk had more oxidative phosphorylation functions. The results could provide further understanding of the unique biological properties of MFGM proteins of goat colostrum and mature milk.

Changes in milk fat globule membrane proteome after pasteurization in human, bovine and caprine species

Milk fat globule membrane (MFGM) proteins have been shown to be very sensitive to processing. This study aims to investigate the thermos-stability of human, bovine, and caprine MFGM proteins after pasteurization. The milk fat globule size was determined using confocal laser scanning microscopy. The MFGM proteins were characterized using SDS-PAGE and label-free proteomic techniques. The results indicated that pasteurization didn’t influence the MFGs size distributions in these three species. A total of 1104, 632, and 137 proteins were identified in human, bovine, and caprine MFGM, respectively. The significantly changed proteins after pasteurization were mainly involved in lipid syntheses and secretion as well as immune response. In addition, the changes of these proteins also differed among species, and a higher thermo-sensitivity was observed in human and caprine MFGM proteins than bovine MFGM proteins. In sum, pasteurization affected MFGM protein composition in different extent among three species.

A proteomic characterization shows differences in the milk fat globule membrane of buffalo and bovine milk

The proteins of the milk fat globule membrane (MFGM) have a number of functions, such as the regulation of milk fat secretion and metabolism, the uptake and transportation of fatty acids in the intestine, and potential protection from bacterial or viral infection. While the proteome of the MFGM in bovine milk has been extensively characterized, knowledge of these proteins in buffalo milk is limited. In this study, a proteomic approach was used to characterize the proteome of the buffalo MFGM. Multiple extraction techniques were used to increase the number of proteins identified, while label free relative quantitative liquid chromatography tandem mass spectrometry was used for comparison between the buffalo and bovine MFGM proteomes. A total of 220 buffalo MFGM proteins and 234 bovine MFGM proteins were identified after being filtered from the initial dataset of 757 and 680 proteins, respectively. A sixfold higher concentration of xanthine oxidoreductase was identified per mass of buffalo MFGM protein extracted, together with significantly greater concentrations of platelet glycoprotein 4, heat shock cognate and calcineurin B homologous protein. The expression of xanthine oxidoreductase in the MFGM of buffalo milk, which can affect milk shelf-life and flavor, was confirmed by Western blot analysis and a heterogeneous distribution of this protein observed in situ on the surface of the MFGM. The high concentration of fat in buffalo milk, together with the differences in the MFGM proteome provide insights into the differences in nutritional profile, biological function and properties of these two milk products.

Effect of the DGAT1 K232A genotype of dairy cows on the milk metabolome and proteome

Diglyceride O-acyltransferase 1 (DGAT1) is the enzyme that catalyzes the synthesis of triglycerides from diglycerides and acyl-coenzyme A. The DGAT1 K232A polymorphism was previously shown to have a significant influence on bovine milk production characteristics (milk yield, protein content, fat content, and fatty acid composition). The mechanism of this influence has, however, not been elucidated. In this study, metabolomics (1H-nuclear magnetic resonance) and proteomics (laser chromatography-tandem mass spectrometry) were applied to determine the serum and lipid metabolite composition and milk fat globule membrane proteome of milk samples from cows with the DGAT1 KK and AA genotypes. The milk samples from cows with the DGAT1 KK genotype contained more stomatin, sphingomyelin, choline, and carnitine, and less citrate, creatine or phosphocreatine, glycerol-phosphocholine, mannose-like sugar, acetyl sugar phosphate, uridine diphosphate (UDP)-related sugar, and orotic acid compared with milk samples from cows with the DGAT1 AA genotype. Based on these results, we propose that the differences between the DGAT1 genotypes may be related to stomatin-sphingomyelin lipid rafts as well as structural (cell membrane) differences in epithelial cells of the mammary gland. In conclusion, our study shows that, in addition to previously described changes in triglyceride composition, cows differing in DGAT1 polymorphism differ in their milk proteome and metabolome, which may help in further understanding the effect of the DGAT1 K232A polymorphism on milk production characteristics.

Proteomic characterization and comparison of mammalian milk fat globule proteomes by iTRAQ analysis.

Milk fat globule membrane (MFGM) proteins have exhibited a relatively larger diversity than other milk fractions, and implicated health beneficial effects. Proteomic analysis of MFGM protein was mainly focused on human, bovine and goat in previous studies. Recently, there is an increasing demand for natural milk from minor dairy animals. Differences in protein components were not yet elucidated that required the integration of this information across multiple species. Thus, iTRAQ analysis of the proteins in MFGM fractions from Holstein, Jersey, yak, buffalo, goat, camel, horse, and human was performed in this study. A total of 520 proteins were identified and quantified in the MFGM fractions. The results were contributed to a comprehensive overview and discriminative profiling of the MFGM proteome across species.

Developmental Changes in the Milk Fat Globule Membrane Proteome During the Transition from Colostrum to Milk

Shotgun proteomics, using amine-reactive isobaric tags (iTRAQ), was used to quantify protein changes in milk fat globule membranes (MFGM) that were isolated from d 1 colostrum and compared with MFGM from d 7 milk. Eight Holstein cows were randomly assigned to 2 groups of 4 cow sample pools for a simple replication of this proteomic analysis using iTRAQ. The iTRAQ labeled peptides from the experiment sample pools were fractionated by strong cation exchange chromatography followed by further fractionation on a microcapillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Data analysis identified 138 bovine proteins in the MFGM with 26 proteins upregulated and 19 proteins downregulated in d 7 MFGM compared with colostrum MFGM. Mucin 1 and 15 were upregulated greater than 7-fold in MFGM from d 7 milk compared with colostrum MFGM. The tripartite complex of proteins of adipophilin, butyrophilin, and xanthine dehydrogenase were individually upregulated in d 7 MFGM 3.4-, 3.2-, and 2.6-fold, respectively, compared with colostrum MFGM. Additional proteins associated with various aspects of lipid transport synthesis and secretion such as acyl-CoA synthetase, lanosterol synthase, lysophosphatidic acid acyltransferase, and fatty acid binding protein were upregulated 2.6- to 5.1-fold in d 7 MFGM compared with colostrum MFGM. In contrast, apolipoproteins A1, C-III, E, and A-IV were downregulated 2.6- to 4.3-fold in d 7 MFGM compared with colostrum MFGM. These data demonstrate that quantitative shotgun proteomics has great potential to provide new insights into mammary development.

Proteome analysis of the bovine milk fat globule: Enhancement of membrane purification

A simple and rapid procedure was developed for the extraction of the milk fat globule membrane from milk removes the large majority of the skim milk proteins for proteome analysis. In order to improve the extraction and the solubilization of the hydrophobic membrane proteins for subsequent two-dimensional gel electrophoresis, four detergents (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, amidosulfobetaine-14, sodium lauroyl sarcosinate and sodium deoxycholate) were tested in the sample preparation, associated with a sonication step. Zwitterionic detergents were shown to be efficient in recovering integral and peripheral proteins from membrane material. Spots were identified by matrix-assisted laser desorption/ionization tandem-time-of-flight (MALDI-TOF/TOF). The advantages of MALDI-TOF/TOF (speed, easiness of analysis, good sensitivity and high mass accuracy) were demonstrated on the milk fat globule membrane proteome investigation. Identified proteins are implicated in a wide range of functions including fat secretion and transport, protein trafficking and regulation.