Human Milk Proteome Research Articles

Regional and longitudinal dynamics of human milk protein components assessed by proteome analysis on a fast and robust micro-flow LC–MS/MS system

An in-depth exploration of molecular composition of human milk could provide a scientific basis for the development of substitutes. The present study was conducted to analyze human milk proteins from 110 individuals from five regions of China and across three stages of lactation to investigate the change patterns. We developed a micro-flow liquid chromatography tandem mass spectrometry (μLC–MS/MS) system with data-independent acquisition (DIA) proteomics technology that can rapidly and stably characterize the human milk proteome. In total, 2796 proteins were identified. Among these proteins, CPM, ACSL1, and RPL13 changed significantly during lactation, and SCP2, GALK1 and GALE changed significantly between regions. Bioinformatics analysis revealed that human milk is altered by complex interactions between genetic and environmental factors. Our results not only reveal the regional and longitudinal patterns of change in human milk proteome but also provide theoretical basis and technical support for the production and quality control of infant formula.

A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or αS1-casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SignificanceDisulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.

Network analysis of the proteome and peptidome sheds light on human milk as a biological system

Proteins and peptides found in human milk have bioactive potential to benefit the newborn and support healthy development. Research has been carried out on the health benefits of proteins and peptides, but many questions still need to be answered about the nature of these components, how they are formed, and how they end up in the milk. This study explored and elucidated the complexity of the human milk proteome and peptidome. Proteins and peptides were analyzed with non-targeted nanoLC-Orbitrap-MS/MS in a selection of 297 milk samples from the CHILD Cohort Study. Protein and peptide abundances were determined, and a network was inferred using Gaussian graphical modeling (GGM), allowing an investigation of direct associations. This study showed that signatures of (1) specific mechanisms of transport of different groups of proteins, (2) proteolytic degradation by proteases and aminopeptidases, and (3) coagulation and complement activation are present in human milk. These results show the value of an integrated approach in evaluating large-scale omics data sets and provide valuable information for studies that aim to associate protein or peptide profiles from biofluids such as milk with specific physiological characteristics.

The human milk proteome and allergy of mother and child: Exploring associations with protein abundances and protein network connectivity.

BackgroundThe human milk proteome comprises a vast number of proteins with immunomodulatory functions, but it is not clear how this relates to allergy of the mother or allergy development in the breastfed infant. This study aimed to explore the relation between the human milk proteome and allergy of both mother and child.MethodsProteins were analyzed in milk samples from a subset of 300 mother-child dyads from the Canadian CHILD Cohort Study, selected based on maternal and child allergy phenotypes. For this selection, the definition of “allergy” included food allergy, eczema, allergic rhinitis, and asthma. Proteins were analyzed with non-targeted shotgun proteomics using filter-aided sample preparation (FASP) and nanoLC-Orbitrap-MS/MS. Protein abundances, based on label-free quantification, were compared using multiple statistical approaches, including univariate, multivariate, and network analyses.ResultsUsing univariate analysis, we observed a trend that milk for infants who develop an allergy by 3 years of age contains higher abundances of immunoglobulin chains, irrespective of the allergy status of the mother. This observation suggests a difference in the milk’s immunological potential, which might be related to the development of the infant’s immune system. Furthermore, network analysis showed overall increased connectivity of proteins in the milk of allergic mothers and milk for infants who ultimately develop an allergy. This difference in connectivity was especially noted for proteins involved in the protein translation machinery and may be due to the physiological status of the mother, which is reflected in the interconnectedness of proteins in her milk. In addition, it was shown that network analysis complements the other methods for data analysis by revealing complex associations between the milk proteome and mother-child allergy status.ConclusionTogether, these findings give new insights into how the human milk proteome, through differences in the abundance of individual proteins and protein-protein associations, relates to the allergy status of mother and child. In addition, these results inspire new research directions into the complex interplay of the mother-milk-infant triad and allergy.

Preterm birth and human milk proteome: are we ready for individualized fortification?

Preterm birth is one of the most pressing clinical problems in obstetrics and neonatology worldwide. One of the most sophisticated components of human milk is the proteome and a better understanding of it can lead to precision guides for feeding preterm infants. In this review, we will examine recent research focused on the human milk proteome and individualized protein fortification of human milk. In both preterm and term birth, the protein content in mother's own milk dropped rapidly in the early postnatal period. Preterm milk had a higher protein content and contained different protein and endogenous peptide compositions compared with term milk. The peptides in gastrointestinal fluids of preterm infants still need further investigation. Individualized fortification is more superior to standard fortification, but it only focuses on the total protein amount. There is no data concerning the composition and posttranslational modifications of proteins and endogenous peptides with fortification and their longterm effects. Comprehensive identification and characterization of the human milk proteome have led to the targeted breast milk fortification theory of preterm infant feeding and has also been enriched by clinical trials. However, to achieve fortification of key proteins and/or endogenous peptides, as standard clinical practice, requires additional studies. Future research should explore the long-term effect of protein fortification and pay more attention to quality rather than quantity in relation to infant body composition and growth outcomes.

Exploring Human Milk Dynamics: Interindividual Variation in Milk Proteome, Peptidome, and Metabolome.

Human milk is a dynamic biofluid, and its detailed composition receives increasing attention. While most studies focus on changes over time or differences between maternal characteristics, interindividual variation receives little attention. Nevertheless, a comprehensive insight into this can help interpret human milk studies and help human milk banks provide targeted milk for recipients. This study aimed to map interindividual variation in the human milk proteome, peptidome, and metabolome and to investigate possible explanations for this variation. A set of 286 milk samples was collected from 29 mothers in the third month postpartum. Samples were pooled per mother, and proteins, peptides, and metabolites were analyzed. A substantial coefficient of variation (>100%) was observed for 4.6% and 36.2% of the proteins and peptides, respectively. In addition, using weighted correlation network analysis (WGCNA), 5 protein and 11 peptide clusters were obtained, showing distinct characteristics. With this, several associations were found between the different data sets and with specific sample characteristics. This study provides insight into the dynamics of human milk protein, peptide, and metabolite composition. In addition, it will support future studies that evaluate the effect size of a parameter of interest by enabling a comparison with natural variability.

Personalized Profiling Reveals Donor- and Lactation-Specific Trends in the Human Milk Proteome and Peptidome

ABSTRACTBackgroundHuman milk is the most genuine form of personalized nutrition, whereby its nutritional and bioactive constituents support the changing needs of the growing infant. Personalized proteome profiling strategies may provide insights into maternal–infant relationships. Proteins and endogenous peptides in human milk play an important role as nutrients for growth and have distinct functionality such as immune defense. Comprehensive monitoring of all of the human milk proteinaceous components, including endogenous peptides, is required to fully understand the changing role of the human milk proteome throughout lactation.ObjectiveWe aimed to investigate the personalized nature of the human milk proteome and peptidome for individual mother–infant dyads.MethodsTwo individual healthy milk donors, aged 29 and 32 y and both of a normal BMI, were longitudinally observed over weeks 1, 2, 3, 4, 6, 8, 10, 12, and 16 postpartum. Milk collection was standardized. Comprehensive variations in the human milk proteinaceous components were assessed using quantitative LC-MS/MS methods.ResultsWe longitudinally profiled the concentrations of >1300 milk proteins and 2000 endogenous milk peptides spanning 16 wk of lactation for 2 individual donors. We observed many gradual and alike changes in both donors related to temporal effects, for instance early lactation was marked by high concentrations of proteins and peptides involved in lactose synthesis and immune development. Uniquely, in 1 of the 2 donors, we observed a substantial anomaly in the milk composition, exclusively at week 6, likely indicating a response to inflammation and/or infection.ConclusionsHere, we provide a resource for characterizing the lactational changes in the human milk proteome, encompassing thousands of proteins and endogenous peptides. Further, we demonstrate the feasibility and benefit of personalized profiling to monitor the influence of milk on the development of the newborn, as well as the health status of each individual mother–infant pair.

Proteomics of Human Milk: Definition of a Discovery Workflow for Clinical Research Studies.

Milk is a complex biological fluid composed mainly of water, carbohydrates, lipids, proteins, and diverse bioactive factors. Human milk represents a unique tailored source of nutrients that adapts during lactation to the specific needs of the developing infant. Proteins in milk have been studied for decades, and proteomics, peptidomics, and glycoproteomics are the main approaches previously deployed to decipher the proteome of human milk. In the present work, we aimed at implementing a highly automated pipeline for the proteomic analysis of human milk with liquid chromatography mass spectrometry (MS). Commercial human milk samples were used to evaluate and optimize workflows. Centrifugation for defatting milk samples was assessed before and after reduction, alkylation, and enzymatic digestion of proteins, without and with presence of surfactants. Skimmed milk samples were analyzed using isobaric labeling-based quantitative MS on an Orbitrap Tribrid mass spectrometer. Sample fractionation using isoelectric focusing was also evaluated to more deeply profile the human milk proteome. Finally, the most appropriate workflow was transferred to a liquid handling workstation for automated sample preparation. In conclusion, we have defined and describe herein an efficient highly automated proteomic workflow for human milk sample analysis. It is compatible with clinical research, possibly allowing the analysis of sufficiently large cohorts of samples.

Human milk proteome: what's new?

The proteome is one of the most complicated and multifunctional components in human milk. Recently, numerous novel characteristics of the human milk proteome have been discovered, which are described and critically examined in this review. Recent human milk proteomics studies have focused on how external factors like geography and environment, or maternal and infant's factors affect the milk proteins, endogenous peptides, their posttransitional modifications (PTMs) and infant utilization. Most of these studies have shown that major protein and endogenous peptide profiles are similar for healthy women and infants. The human milk proteome has been expanded by providing novel insights into PTMs like glycosylation and phosphorylation, and how the proteins and peptides are digested and utilized by the infant. All human milk proteomics studies are subject to conditions in which the samples were collected, handled and stored. Significant technological advancements in mass spectrometry have considerably enabled a deeper and more comprehensive identification and characterization of the expanded human milk proteome. However, data concerning human milk from mothers with infections or illnesses and mothers nursing more vulnerable infants are still limited and the roles of the components of the human milk proteome have not yet been sufficiently elucidated.

Systematic examination of protein extraction, proteolytic glycopeptide enrichment and MS/MS fragmentation techniques for site-specific profiling of human milk N-glycoproteins

Human milk contains numerous N-glycoproteins with functions that provide protection to the infant. Increasing understanding of the functional role of human milk glycoproteins within the infant requires toolsets to comprehensively profile their site-specific glycosylation patterns. However, optimized methods for site-specific glycosylation analysis across the entire human milk proteome are not available. Therefore, we performed a systematic analysis of techniques for profiling the sites and compositions of N-glycans in human milk using liquid chromatography/mass spectrometry. To decrease interference from non-target molecules, we compared techniques for protein extraction, including ethanol (EtOH) precipitation, trichloroacetic acid precipitation, molecular weight cut-off filtration and techniques for tryptic glycopeptide enrichment, including C18-, porous graphitized carbon and hydrophilic interaction liquid chromatography (HILIC)-solid phase extraction (SPE) and acetone precipitation. We compared the capacity of higher-energy collision dissociation, electron-transfer dissociation and electron-transfer/higher-energy collision dissociation (EThcD) to produce fragment ions that would enable effective identification of the glycan composition, peptide sequence and glycosylation site. Of these methods, a combination of EtOH precipitation, HILIC-SPE and EThcD-fragmentation was the most effective for human milk N-glycopeptide profiling. This optimized approach significantly increased the number of N-glycopeptides and precursor N-glycoproteins (246 N-glycopeptides from 29 glycoproteins) compared with a more common extraction approach with no protein extraction and C18 clean-up (62 N-glycopeptides from 11 glycoproteins). The advancement in methods for human milk N-glycoproteins provided by this study represents a key step for better understanding the function of glycoproteins within the breast milk-fed infant.

The Functional Power of the Human Milk Proteome.

Human milk is the most complete and ideal form of nutrition for the developing infant. The composition of human milk consistently changes throughout lactation to meet the changing functional needs of the infant. The human milk proteome is an essential milk component consisting of proteins, including enzymes/proteases, glycoproteins, and endogenous peptides. These compounds may contribute to the healthy development in a synergistic way by affecting growth, maturation of the immune system, from innate to adaptive immunity, and the gut. A comprehensive overview of the human milk proteome, covering all of its components, is lacking, even though numerous analyses of human milk proteins have been reported. Such data could substantially aid in our understanding of the functionality of each constituent of the proteome. This review will highlight each of the aforementioned components of human milk and emphasize the functionality of the proteome throughout lactation, including nutrient delivery and enhanced bioavailability of nutrients for growth, cognitive development, immune defense, and gut maturation.

Excretion of Dietary Cow's Milk Derived Peptides Into Breast Milk.

Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow's milk-derived β-Lg f(125–135) and β-casein f(81–92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-β-Lg antibody, but dot-blot analysis confirmed the occurrence of β-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.

Investigation of human milk proteins by proteomics

Human milk is the optimal food for infant nutrition and growth. Proteins are abundant and represent a key element in human milk. With recent developments in proteomics, more tools are available to explore human milk proteins. This article aims to review the recent investigations of human milk proteins using proteomic methodologies. This review focuses on using proteomics as a tool to study the components of human milk proteins; dynamics of human milk proteins during lactation; comparison of proteome from human milk and other source milk, phosphoprotein and glycoprotein analysis of human milk; endogenous peptides in human milk; and the human milk proteome and its correlation to curing of various diseases. Proteomics technology has enabled the study of human milk proteins in the era of micronutrient research, and the results of these studies will be helpful for further analysis of mother and infant health.

Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis.

This work describes a simple sample pretreatment method for the fractionation of human milk proteins (into their two main groups, whey and caseins) prior to their analysis. The protein-extraction protocol is based on the addition of calcium phosphate to nonadjusted pH human milk. The combination of calcium ions with phosphate results in an effective coprecipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), LC-MS/MS, and capillary electrophoresis (CE) analysis. The results evidence a significant decrease in contamination of casein fraction with whey proteins and vice versa compared with the conventional isoelectric precipitation of caseins. In addition, CE fraction collection coupled to LC-MS/MS (off-line coupling) has been successfully applied to the identification of minor proteins in this complex matrix. The methodology presented here constitutes a promising tool to enlarge the knowledge of human milk proteome.

An interactomics overview of the human and bovine milk proteome over lactation.

BackgroundMilk is the most important food for growth and development of the neonate, because of its nutrient composition and presence of many bioactive proteins. Differences between human and bovine milk in low abundant proteins have not been extensively studied. To better understand the differences between human and bovine milk, the qualitative and quantitative differences in the milk proteome as well as their changes over lactation were compared using both label-free and labelled proteomics techniques. These datasets were analysed and compared, to better understand the role of milk proteins in development of the newborn.MethodsHuman and bovine milk samples were prepared by using filter-aided sample preparation (FASP) combined with dimethyl labelling and analysed by nano LC LTQ-Orbitrap XL mass spectrometry.ResultsThe human and bovine milk proteome show similarities with regard to the distribution over biological functions, especially the dominant presence of enzymes, transport and immune-related proteins. At a quantitative level, the human and bovine milk proteome differed not only between species but also over lactation within species. Dominant enzymes that differed between species were those assisting in nutrient digestion, with bile salt-activated lipase being abundant in human milk and pancreatic ribonuclease being abundant in bovine milk. As lactation advances, immune-related proteins decreased slower in human milk compared to bovine milk. Notwithstanding these quantitative differences, analysis of human and bovine co-expression networks and protein-protein interaction networks indicated that a subset of milk proteins displayed highly similar interactions in each of the different networks, which may be related to the general importance of milk in nutrition and healthy development of the newborn.ConclusionsOur findings promote a better understanding of the differences and similarities in dynamics of human and bovine milk proteins, thereby also providing guidance for further improvement of infant formula.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0110-0) contains supplementary material, which is available to authorized users.

The Effect of Genetic Variation on the Human Milk Proteome

Milk proteins provide more than amino acids to offspring. Many milk proteins have been shown to have other biological roles such as digestion, nutrient absorption, and immunodevelopment. We previously catalogued proteins in human milk by LC‐MS/MS using a reference database for the peptide spectrum matching (PSM). However, proteins can only be identified via PSM if the sequence of the protein is present in the database. To address this problem, we used RNA‐sequencing to develop a comprehensive database of protein sequences for milk samples from three subjects with different ethnicities: Caucasian, African American, and Hispanic. We then used these databases to search the spectra generated from LC‐MS/MS analysis of whole milk samples from the same three subjects. Using these custom proteome databases, we identified 85 milk proteins with genetic variation relative to the reference sequence. These 85 milk proteins contained 184 single amino acid polymorphisms and one insertion. Importantly, these genetic variants include common isoforms of the major proteins in milk such as the caseins and lactoferrin. Based on exclusive spectrum counts, some of the variants identified are more common in milk than the protein sequence derived from the reference database. Examination of the exclusive spectrum counts also demonstrates genetic differences between the milk from these subjects of different ethnicities. The presence of different isoforms of the major milk proteins may have biological consequences for offspring. Our study also suggests that genetic differences should be considered when comparing milk proteomes between different human populations.Support or Funding InformationFunded in part by California National Primate Research Center Postdoc Pilot Grant to D.L., supplement to NIH/NCRR P51 RR00169, K.B. was supported in part by a fellowship under the Training Program in Biomolecular Technology (NIGMS‐NIH T32‐GM008799) at the University of California, Davis.

Spatiotemporal Profile of the Human Milk Proteome across Populations and Lactation Stages: GEHM Study Insights

Improved understanding of temporal and geographical differences in the human milk proteome may provide novel insights into changes in milk composition and function across the globe during breastfeeding. We have compared milk proteomes among three populations (Shanghai, China, Mexico City, Mexico and Cincinnati, United States; n = 9 for each population) at two different stages of lactation (4 and 26 weeks), with all samples collected as part of the Global Exploration of Human Milk (GEHM) study. At each of the two lactation stages, the profiles of milk proteins are largely similar among the three populations. Differences were found to be greater with respect to lactation stage as opposed to population. Interestingly, proteins with significant abundance changes over time show patterns of regulation that are broadly similar among populations. Nevertheless, at each of the two lactation stages, immunoglobulins IgA2, IgG1, IgG2 and IgM are present at higher levels in milk from China and Mexico compared to those from the U.S. with fold changes between China and U.S. samples of 2.0, 1.6, 2.3 and 2.1, respectively, and fold changes between Mexico and U.S. samples of1.8, 1.3, 1.1 and 2.0, respectively. Similar observations can be made for the antimicrobial protein lactoferrin. Our findings suggest broad congruency in milk protein composition among the three populations. This study also suggests that a number of host‐defense proteins may be more abundant in human milk from developing countries. This research was supported by Mead Johnson Pediatric Nutrition Institute.

Proteomics as a tool to explore human milk in health and disease

Proteins in milk have wide range of functions, they are carriers of minerals or chemically vulnerable and insoluble vitamins and other compounds, stabilisers of large aggregates or micelles of lipids, and components of both innate and acquired immune defence systems. Together with other components of milk, proteins may also contribute to the selection and establishment of appropriate microbiome in the gut of the infant. The proteome of mammalian milk is now known to be dynamic and changes radically with time after birth from colostrum to mature lactation. Significantly, immune and innate defence proteins appear in milk during infection of the mammary gland and possibly also during systemic infections. The understanding of the human milk proteome and how it changes with time during lactation and in disease is developing rapidly, and is to a large extent informed by proteomics of the milks of non-human mammals, domestic animals in particular. We review general methods now being applied for proteomic analysis of human milk. Moreover we place emphasis on how the milk proteome may change in different ways in response to disease, mastitis in particular, how such changes may be specific to pathogen types, and we give some insights about evolution.

Time‐resolved quantitative analysis of human milk proteome reveals developing milk and mammary‐gland functions during the first year of lactation

In‐depth understanding of the developing function of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of HM proteome. We analyzed the HM proteome (n =10 women with samples at 1, 4, 12, 24, 36 and 52 weeks) using a quantitative proteomic approach. 1147 proteins were identified with 580 being quantified. Principle component analysis showed that HM proteomes transition throughout the first year of lactation. Abundance changes of IgG, sIgA and IgM display distinct features during the first year. Complement components and other acute‐phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that milk fatty‐acid (FA) metabolism shifts from more direct blood intake to more de novo mammary synthesis, which may alter the lipoprotein profile in lactating women and the composition of milk FAs. Glycoprotein metabolism transitions to catabolism in late lactation, which may increase the presence of free anti‐pathogenic HM oligosaccharides and peptides. Increased metabolic activity in late lactation can lead to a larger amount of deleterious by‐products. Cellular detoxification machinery is also transformed, thereby accommodating increased metabolic activities in late lactation. The multiple functions of the HM proteins become more apparent from this study.

Temporal Changes in Milk Proteomes Reveal Developing Milk Functions

Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.