Secretory membranes of the lactating mammary gland

Summary The lactating mammary gland is one of the most highly differentiated and metabolically active organs in the body. Membranes of the lactating mammary cell have important roles in transmitting from one membrane to another of hormonal information and in milk secretion, which is the final event. During milk secretion, the projection of the surface membrane into the alveolar lumen by enveloping intracellular lipid droplets with the apical plasma membrane is one of the most remarkable aspects of biological membrane action throughout nature.This review focuses on current knowledge about membranes in the lactating mammary gland. (1) Advances in the isolation and properties of membranes, especially the plasma membrane and Golgi-derived secretory vesicles, concerned with milk secretion from the lactating mammary gland are described. (2) Milk serum components are secreted by fusing the membranes of secretory vesicles that condense milk secretions with the plasma membrane in the apical regions. This occurs through the formation of a tubular-shaped projection and vesicular depression in a ball-and-socket configuration, as well as by simple fusion. (3) Intracellular lipid droplets are directly extruded from the mammary epithelial cells by progressive envelopment of the plasma membranes in the apical regions. (4) The balance between the surface volume lost in enveloping lipid droplets and that provided by fusion of the secretory vesicle and other vesicles with the apical plasma membrane is discussed. (5) The membrane surrounding a milk fat globule, which is referred to as the milk fat globule membrane (MFGM), is composed of at least the coating membrane of an intracellular lipid droplet, of the apical plasma membrane and secretory vesicle membrane, and of a coat material. Consequently, MFGM is molecularly different from the plasma membrane in composition. (6) MFGM of bovine milk is structurally composed of an inner coating membrane and outer plasma membrane just after segregation. These two membranes are fused and reorganized through a process of vesiculation and fragmentation to stabilize the fat globules. Hypothetical structural models for MFGM from bovine milk fat globules just after secretion and after rearrangement are proposed.Abbrevations MFGM milk fat globule membrane - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - INT 2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - Sph sphingomyelin - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PS phosphatidyl serine - PI phosphatidyl inositol - PAS periodic acid-Schiff reagent - CB Coomassie brilliant blue R-250 Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

A Test of Current Models for the Mechanism of Milk‐Lipid Droplet Secretion

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin‐2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N‐linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN‐enhanced green fluorescent protein was highly mobile in areas of mouse milk‐lipid droplets that had not undergone post‐secretion changes, and endogenous mouse BTN comprised only 0.5–0.7% (w/w) of the total protein, i.e. over 50‐fold less than in the milk‐lipid droplets of cow and other species. These data are incompatible with models of milk‐lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk .     

Freeze-fracture observations of the lactating rat mammary gland. Membrane events during milk fat secretion

Membrane events during milk fat secretion were analyzed by freeze-fracture of the rat mammary gland. Two modes of milk fat secretion were observed: extrusion of fat droplets surrounded by a portion of the apical plasma membrane of the alveolar epithelial cells and, less frequently, release into the alveolar lumen of fat droplets contained in intracytoplasmic vacuoles. The extrusion process consists of two asynchronous events: clearing of membrane particles (probably including integral membrane proteins) and bulging of the apical plasma membrane. Most fat droplets are extruded with a bilayer membrane envelope (milk fat globule membrane) partially devoid of particles. The segregation of membrane particles may represent the onset of a process of structural degradation of the milk fat globule membrane.     

Milk Fat Content and DGAT1 Genotype Determine Lipid Composition of the Milk Fat Globule Membrane

During secretion of milk fat globules, triacylglycerol (TAG) droplets are enveloped by a phospholipid (PL) trilayer. Globule size has been found to be related to polar lipid composition and fat content, and milk fat content and fatty acid composition have been associated with the diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism; however, the association between the DGAT1 polymorphism and fat globule size and polar lipid composition has not been studied. The ratio between polar and neutral lipids as well as the composition of the polar lipids in milk has industrial as well as nutritional and health implications. Understanding phenotypic and genotypic factors influencing these parameters could contribute to improving milk lipid composition for dairy products. The focus of the present study was to determine the effect of both fat content and DGAT1 polymorphism on PL/TAG ratio, as a marker for milk fat globule size, and detailed PL composition. Milk samples were selected from 200 cows such that there were equal numbers of samples for the different fat contents as well as per DGAT1 genotype. Samples were analyzed for neutral and polar lipid concentration and composition. PL/TAG ratio was significantly associated with both fat content and DGAT1 genotype. Phosphatidylinositol and phosphatidylserine concentrations were associated with fat content*DGAT1 genotype with a stronger association for the AA than the KK genotype. Sphingomyelin concentration tended to interact with fat content*DGAT1 genotype. Phosphatidylethanolamine (PE) concentration showed a biphasic response to fat content, suggesting that multiple biological processes influence its concentration. These results provide a new direction for controlling polar lipid concentration and composition in milk through selective breeding of cows.     

Butyrophilin of milk lipid globule membrane contains N-linked carbohydrates and cross-links with xanthine oxidase

1. N-glycanase, but not O-glycanase, released carbohydrates from butyrophilin of rat and cow milk lipid globule membranes. 2. 1-Deoxynojirimycin, and inhibitor of glucosidases I and II of the glycoprotein processing pathway, increased the amount or extent of glycosylation of butyrophilin in rat milk lipid globules. 3. Butyrophilin and xanthine oxidase of milk lipid globule membrane had a nearest neighbor relationship, as demonstrated through specific crosslinking of these proteins. 4. From these results it is suggested that butyrophilin has asparagine-linked oligosaccharides which bypass the processing apparatus of endoplasmic reticulum and Golgi apparatus. Butyrophilin may be responsible for anchoring xanthine oxidase to the inner (cytoplasmic) face of milk lipid globule membrane.     

BIOCHEMICAL AND MORPHOLOGICAL COMPARISON OF PLASMA MEMBRANE AND MILK FAT GLOBULE MEMBRANE FROM BOVINE MAMMARY GLAND

Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, β-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.     

Intracellular origin and secretion of milk fat globules

The cream or fat fraction of milk consists of fat droplets composed primarily of triacylglycerols that are surrounded by cellular membranes. In this review we discuss what is known about how these droplets are formed in and secreted by mammary epithelial cells during lactation. This secretion mechanism, which appears to be unique, is unlike the exocytotic mechanism used by other cell types to secrete lipids. Milk fat globules originate as small, triacylglycerol-rich, droplets that are formed on or in endoplasmic reticulum membranes. These droplets are released from endoplasmic reticulum into the cytosol as microlipid droplets coated by proteins and polar lipids. Microlipid droplets can fuse with each other to form larger cytoplasmic lipid droplets. Droplets of all sizes appear to be unidirectionally transported to apical cell regions by as yet unknown mechanisms that may involve cytoskeletal elements. These lipid droplets appear to be secreted from the cell in which they were formed by being progressively enveloped in differentiated regions of apical plasma membrane. While plasma membrane envelopment appears to be the primary mechanism by which lipid droplets are released from the cell, a mechanism involving exocytosis of lipid droplets from cytoplasmic vacuoles also has been described. As discussed herein, while we have a general overview of the steps leading to the fat globules of milk, virtually nothing is known about the molecular mechanisms involved in milk fat globule formation, intracellular transit, and secretion.     

Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells

Milk lipid globules of various species are surrounded by a membrane structure that is separated from the triglyceride core of the globule by a densely staining fuzzy coat layer of 10- to 50-nm thickness. This internal coat structure remains attached to the membrane during isolation and extraction with low- and high-salt buffers, is insoluble in nondenaturing detergents, and is enriched in an acidic glycoprotein (butyrophilin) with an apparent Mr of 67,000. Guinea pig antibodies against this protein, which show cross-reaction with the corresponding protein in some (goat) but not other (human, rat) species, have been used for localization of butyrophilin on frozen sections of various tissues from cow by immunofluorescence and electron microscopy. Significant reaction is found only in milk-secreting epithelial cells and not in other cell types of mammary gland and various epithelial tissues. In milk-secreting cells, the staining is restricted to the apical cell surface, including budding milk lipid globules, and to the periphery of the milk lipid globules contained in the alveolar lumina. These findings indicate that butyrophilin, which is constitutively secreted by surface budding in coordination with milk lipid production, is located at the apical surface and is not detected at basolateral surfaces, in endoplasmic reticulum, and in Golgi apparatus. This protein structure represents an example of a cell type-specific cytoskeletal component in a cell apex. It is suggested that this antigen provides a specific marker for the apical surface of milk- secreting cells and that butyrophilin is involved in the vectorial discharge of milk lipid globules.     

Relationships between milks differentiated on native milk fat globule characteristics and fat,protein and calcium compositions

Many studies have shown that milk fat globule (MFG) diameter varies in dairy cows in relation to diet and/or breed. However, the mechanisms governing the size of the fat globules remain hypothetical. Our objective was to determine the variable biochemical characteristics (fat, protein, fatty acids (FA), casein and calcium (Ca) contents) between individual milk which differed in both MFG diameter and membrane content, in order to speculate about the links between milk synthesis and MFG secretion. With this aim, we built five databases of individual milk samples from 21 experiments performed between 2003 and 2011. Three of them grouped data from trials dealing with breed/diet effects and included information about: (i) MFG size/membrane, fat and protein contents (n=982), (ii) previous parameters plus FA profile (n=529) and (iii) previous parameters plus true protein composition and calcium contents (n=101). A hierarchical clustering analysis performed on these three databases yielded four groups differing in the MFG characteristics. We observed significant differences among groups for the following parameters: (i) fat content and fat : protein ratio; (ii) de novo and polyunsaturated FA contents; (iii) Ca contents. These relationships could result from potential process regulating the synthesis and secretion of MFG: (i) the apical membrane turnover for MFG secretion and (ii) cytoplasmic lipid droplet formation in the lactocyte during its migration from the basal to the apical pole. The two other databases grouped data from trials dealing with milking frequency (n=211), milking kinetics and milk type (residual v. cisternal) (n=224). They were used to study the relationships between the size of the MFG and milk composition for high native fat contents (from 60 up to 100 g/kg in residual milks). We observed curvilinear relationships between the size of the MFG and fat content, as well as with the fat : protein ratio. This result suggests that MFG diameter reaches a threshold but mechanisms are still unknown.     

The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis.

Functional relations between exocytotic vesicle membranes, plasmalemma and milk fat globule membranes (MFGM) were studied during the final stages of mouse mammary gland differentiation, in the gland during full lactation and in the postpartum gland in which the synthesis of secretory products was partly inhibited by application of 2-Br-alpha-ergocryptine. Analysis of ultrathin sections, freeze-fracture replicas, scanning electron microscopy and application of a cytochemical marker filipin showed that the apocrine secretion of lipid globules was closely related to the exocytosis of milk proteins. During the last days of gestation the secretion of lipid globules resulted from many exocytotic events of the secretory vesicles that accumulated and fused around the cytoplasmic lipid droplets. Seldom the lipid droplet protruded partly into the gland lumen and a part of its surface became covered with the apical plasmalemma. Although apical plasmalemma became more important in the formation of MFGM in the postpartum period, we could still confirm a direct contribution of secretory vesicle membranes to the final detachment of the lipid globule. The application of 2-Br-alpha-ergocryptine hindered the apocrine secretion of the lipid globules and a situation similar to the situation in the prepartum gland was observed.     

Butyrophilin and xanthine oxidase occur in constant molar proportions in milk lipid globule membrane but vary in amount with breed and stage of lactation

Summary Butyrophilin and xanthine oxidase, major proteins of milk lipid globule membrane, both accounted for significantly higher percentages of total protein in membrane samples from Holstein than from Jersey animals. Both were high in membranes from animals in early lactation, both decreased in amount as lactation progressed to the midpoint, and then both rose in amount toward the end of lactation. In samples from both Holstein and Jersey animals, butyrophilin and xanthine oxidase were present in constant molar proportions of about 41. These proteins co-enriched together with low molecular weight GTP-binding proteins in a high salt and nonionic detergent insoluble fraction of milk lipid globule membrane. Butyrophilin and xanthine oxidase content of membranes was not related to milk lipid globule diameter, suggesting that these proteins alone may not be involved solely in anchoring the membrane to the lipid globule surface. However, the possibility that a complex composed in part of butyrophilin and xanthine oxidase serves an anchoring function remains a possibility.     

Phosphoproteomics of the goat milk fat globule membrane: New insights into lipid droplet secretion from the mammary epithelial cell

Mechanisms of milk lipid secretion are highly controversial. Analyzing the fine protein composition of the “milk fat globule membrane” (MFGM), the triple‐layered membrane surrounding milk lipid droplets (LDs) can provide mechanistic clues to better understand LD biosynthesis and secretion pathways in mammary epithelial cells (MECs). We therefore combined a high‐sensitive Q‐Exactive LC‐MS/MS analysis of MFGM‐derived peptides to the use of an in‐house database intended to improve protein identification in the goat species. Using this approach, we performed the identification of 442 functional groups of proteins in the MFGM from goat milk. To get a more dynamic view of intracellular mechanisms driving LD dynamics in the MECs, we decided to investigate for the first time whether MFGM proteins were phosphorylated. MFGM proteins were sequentially digested by lysine‐C and trypsin proteases and the resulting peptides were fractionated by a strong cation exchange chromatography. Titanium beads were used to enrich phosphopeptides from strong cation exchange chromatography eluted fractions. This approach lets us pinpoint 271 sites of phosphorylation on 124 unique goat MFGM proteins. Enriched GO terms associated with phosphorylated MFGM proteins were protein transport and actin cytoskeleton organization. Gained data are discussed with regard to lipid secretory mechanisms in the MECs. All MS data have been deposited in the ProteomeXchange with identifier PXD001039 ( http://proteomecentral.proteomexchange.org/dataset/PXD001039 ).     

Bovine milk exosome proteome

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.     

Immunocytochemical Evidence for Golgi Vesicle Involvement in Milk Fat Globule Secretion

The exact mechanism of secretion of the milk fat globule (MFG) from the mammary secretory cell is still controversial. We have previously suggested close involvement of Golgi vesicles in this process. This paper provides direct immunocytochemical evidence that butyrophilin is present in the Golgi stack and vesicles in ovine and caprine mammary glands. We suggest that it is the butyrophilin in the Golgi vesicle membrane that forms the specific association with the adipophilin on the lipid surface in the cytoplasm. Exocytosis of the associated Golgi vesicle will then initiate the process of MFG secretion. Further exocytosis of associated Golgi vesicles will continue and complete the process. Areas of the plasmalemma that have butyrophilin delivered by previous non-lipid associated Golgi exocytoses may also contribute to the process of forming the milk fat globule membrane (MFGM).     

The adipophilin C terminus is a self-folding membrane-binding domain that is important for milk lipid secretion

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.     

Differential scanning calorimetry of milk fat globule membranes

Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned.     

Milk fat globule membranes devoid of intramembranous particles

When isolated milk fat globule membranes from bovine, human, and murine (rat) milk were examined by freeze-fracturing most of the membrane faces were devoid of membrane-intercalated particles whereas a minor portion showed relatively few particles, either in clusters or in apparent random distribution. A reduced particle density was also noted in membranes of intra-alveolar milk fat globules of cows and rats, in contrast to high particle densities in the apical plasma membrane of lactating epithelial cells. The observations suggest that certain membrane constituents recognized as intramembranous particles either are displaced from the region of the apical surface of the mammary epithelial cell which is involved in milk fat globule budding or are dislocated and rearranged during the budding process.     

Regulation and functional relevance of milk fat globules and their components in the mammary gland

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.     

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.     

Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins

Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as environmental factors. It is therefore important to study the qualitative and quantitative differences of bovine milk samples. Proteins in milk can be present in casein micelles, in the serum (the water soluble fraction) or in fat globules. These fat globules have a double membrane layer with proteins being bound to or being incapsulated in the membrane layer. The identification and molecular composition of the milk proteins have gained increased interest in recent years. Proteomic techniques make it now possible to identify up to many thousands of proteins in one sample, however quantification of proteins is as yet not straightforward. We analyzed the proteins of the milk fat globule membrane using dimethyl labeling methods combined with a filter-aided sample preparation protocol. Using these methods, it is now possible to quantitatively study the detailed protein composition of many milk samples in a short period of time.