Identification of common and distinct origins of human serum and breastmilk IgA1 by mass spectrometry-based clonal profiling.

The most abundant immunoglobulin present in the human body is IgA. It has the highest concentrations at the mucosal lining and in biofluids such as milk and is the second most abundant class of antibodies in serum. We assessed the structural diversity and clonal repertoire of IgA1-containing molecular assemblies longitudinally in human serum and milk from three donors using a mass spectrometry-based approach. IgA-containing molecules purified from serum or milk were assessed by the release and subsequent analysis of their Fab fragments. Our data revealed that serum IgA1 consists of two distinct structural populations, namely monomeric IgA1 (∼80%) and dimeric joining (J-) chain coupled IgA1 (∼20%). Also, we confirmed that IgA1 in milk is present solely as secretory (S)IgA, consisting of two (∼50%), three (∼33%) or four (∼17%) IgA1 molecules assembled with a J-chain and secretory component (SC). Interestingly, the serum and milk IgA1-Fab repertoires were distinct between monomeric, and J-chain coupled dimeric IgA1. The serum dimeric J-chain coupled IgA1 repertoire contained several abundant clones also observed in the milk IgA1 repertoire. The latter repertoire had little to no overlap with the serum monomeric IgA1 repertoire. This suggests that human IgA1s have (at least) two distinct origins; one of these produces dimeric J-chain coupled IgA1 molecules, shared in human serum and milk, and another produces monomeric IgA1 ending up exclusively in serum.

A case series exploring the human milk polyclonal IgA1 response to repeated SARS-CoV-2 vaccinations by LC-MS based fab profiling.

Introduction: Upon vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humans will start to produce antibodies targeting virus specific antigens that will end up in circulation. In lactating women such antibodies will also end up in breastmilk, primarily in the form of secretory immunoglobulin A1 (SIgA1), the most abundant immunoglobulin (Ig) in human milk. Here we set out to investigate the SIgA1 clonal repertoire response to repeated SARS-CoV-2 vaccination, using a LC-MS fragment antigen-binding (Fab) clonal profiling approach. Methods: We analyzed the breastmilk of six donors from a larger cohort of 109 lactating mothers who received one of three commonly used SARS-CoV-2 vaccines. We quantitatively monitored the SIgA1 Fab clonal profile over 16 timepoints, from just prior to the first vaccination until 15 days after the second vaccination. Results: In all donors, we detected a population of 89-191 vaccine induced clones. These populations were unique to each donor and heterogeneous with respect to individual clonal concentrations, total clonal titer, and population size. The vaccine induced clones were dominated by persistent clones (68%) which came up after the first vaccination and were retained or reoccurred after the second vaccination. However, we also observe transient SIgA1 clones (16%) which dissipated before the second vaccination, and vaccine induced clones which uniquely emerged only after the second vaccination (16%). These distinct populations were observed in all analyzed donors, regardless of the administered vaccine. Discussion: Our findings suggest that while individual donors have highly unique human milk SIgA1 clonal profiles and a highly personalized SIgA1 response to SARS-CoV-2 vaccination, there are also commonalities in vaccine induced responses.

Proteoform Profiles Reveal That Alpha-1-Antitrypsin in Human Serum and Milk Is Derived From a Common Source.

The Alpha-1-Antitrypsin (A1AT) protein is an important protease inhibitor highly abundant in human serum and other body fluids. Additional to functioning as a protease inhibitor, A1AT is an important acute phase protein. Here, we set out to compare the proteoform profiles of A1AT purified from the human serum and milk of eight healthy donors to determine the origin of human milk A1AT. Following affinity purification, size-exclusion chromatography coupled to native mass spectrometry was used to monitor individual proteoform profiles comparing inter- and intra-donor profiles. The A1AT intra-donor proteoform profiles were found to be highly identical between serum and milk, while they were highly distinct between donors, even when comparing only serum or milk samples. The observed inter-donor proteoform variability was due to differences in the abundances of different N-glycoforms, mainly due to branching, fucosylation, and the relative abundance of N-terminally processed A1AT fragments. From our data we conclude that nearly all A1AT in serum and milk is synthesized by a common source, i.e. the liver, and then secreted into the circulation and enters the mammary gland via diffusion or transport. Thereby, proteoform profile changes, as seen upon infection and/or inflammation in the blood will be reflected in the milk, which may then be transferred to the breastfed infant.

A Direct MS-Based Approach to Profile Human Milk Secretory Immunoglobulin A (IgA1) Reveals Donor-Specific Clonal Repertoires With High Longitudinal Stability.

Recently, a mass spectrometry-based approach was introduced to directly assess the IgG1 immunoglobulin clonal repertoires in plasma. Here we expanded upon this approach by describing a mass spectrometry-based technique to assess specifically the clonal repertoire of another important class of immunoglobulin molecules, IgA1, and show it is efficiently and robustly applicable to either milk or plasma samples. Focusing on two individual healthy donors, whose milk was sampled longitudinally during the first 16 weeks of lactation, we demonstrate that the total repertoire of milk sIgA1 is dominated by only 50-500 clones, even though the human body theoretically can generate several orders of magnitude more clones. We show that in each donor the sIgA1 repertoire only changes marginally and quite gradually over the monitored 16-week period of lactation. Furthermore, the observed overlap in clonal repertoires between the two individual donors is close to non-existent. Mothers provide protection to their newborn infants directly by the transfer of antibodies via breastfeeding. The approach introduced here, can be used to visualize the clonal repertoire transferred from mother to infant and to detect changes in-time in that repertoire adapting to changes in maternal physiology.

Monitoring Human Milk ß-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome.

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant's best start at a healthy life. One key component of human milk is ß-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk's endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of ß-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of ß-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in ß-casein's known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of ß-casein may be important as it resides on known ß-casein-derived antimicrobial peptide sequences.

Human Milk from Previously COVID-19-Infected Mothers: The Effect of Pasteurization on Specific Antibodies and Neutralization Capacity.

BACKGROUND: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. METHODS: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). RESULTS: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. CONCLUSIONS: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Optimization of a human milk-directed quantitative sIgA ELISA method substantiated by mass spectrometry.

Immunoglobulins are the primary protective products in human milk and are responsible for transferring maternal pathogen memory to the infant, providing protection by binding to recognized pathogens and inhibiting virulence. To better understand potentially protective/anti-infective compounds in human milk, the establishment of human milk-tailored analytical approaches is crucial, as most contemporary analytical methods have been optimized for plasma or serum. One of the most prominent immunoglobulins in human milk is secretory immunoglobulin A (sIgA), which may be relevant for the protection of breastfed infants from harmful pathogens. Advanced sIgA detection methods can help monitor the immune status and development of the mother-infant dyad. We therefore developed an enzyme-linked immunosorbent assay (ELISA) sIgA method for the quantitative analysis of IgA plus secretory component (SC), validated with sIgA standards and substantiated by mass spectrometry (MS)-based proteomics. A very strong correlation was observed between the MS-detected IgA1 and the human milk-specific sIgA ELISA (r = 0.82). Overall, the MS data indicate that the developed human milk sIgA ELISA does not differentiate between sIgA1 and sIgA2 and is, therefore, a reflection of total sIgA. Furthermore, our MS data and the human milk-derived sIgA ELISA data are better correlated than data derived from a standard serum IgA ELISA kit (relative to MS IgA1 r = 0.82 and r = 0.42, respectively). We therefore propose our human milk-specific sIgA ELISA as an ideal quantitative indicator of total sIgA with advantages over current serum IgA ELISA kits.

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

BACKGROUND: Human 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. OBJECTIVE: We aimed to investigate the personalized nature of the human milk proteome and peptidome for individual mother-infant dyads. METHODS: Two 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. RESULTS: We 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. CONCLUSIONS: Here, 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.

Human milk proteome: what's new?

PURPOSE OF REVIEW: 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 FINDINGS: 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. SUMMARY: 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.

Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes.

Protein N-glycosylation on human milk proteins assists in protecting an infant's health and functions among others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother's milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein- and N-site-specific quantitative information. Here, we describe an automated platform using hydrophilic-interaction chromatography (HILIC)-based cartridges enabling the proteome-wide monitoring of intact N-glycopeptides using just a digest of 150 µg of breast milk protein. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. Although each glycoprotein, N-glycosylation site, and attached glycan revealed distinct dynamic changes, we did observe a few general trends. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Building on the improved glycoproteomics approach outlined above, future studies are warranted to reveal the potential impact of the observed glycosylation microheterogeneity on the healthy development of infants.

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.

A Randomized Trial of Maternal Docosahexaenoic Acid Supplementation to Reduce Inflammation in Extremely Preterm Infants.

Maternal supplementation with 1000 mg/day docosahexaenoic acid (DHA) provides third trimester DHA accretion levels in breast milk for the preterm infant. We hypothesized that DHA supplementation to mothers providing breastmilk for extremely preterm infants would result in decreased inflammatory markers, in the infant. Mother/infant dyads (n = 27) were enrolled at birth and mothers were assigned to receive 200 or 1000 mg/day of DHA. Milk and plasma samples were analyzed for fatty acids and inflammatory markers. Decreases in inflammation were observed in both maternal and infant plasma and correlated with red blood cell (RBC) DHA levels. The fact that maternal DHA supplementation decreases infant markers of inflammation implies that DHA, delivered through breastmilk, has the potential to decrease inflammation in the infant.

Toward an efficient workflow for the analysis of the human milk peptidome.

There is a growing interest for investigating endogenous peptides from human biofluids which may provide yet unknown functional benefits or provide an early indication of disease states as potential biomarkers. A major technical bottleneck in the investigation of endogenous peptides from body fluids, e.g., serum, urine, saliva, and milk, is that each of these fluids seems to require unique workflows for peptide extraction and analysis. Thus, protocols optimized for serum cannot be directly translated to milk. One biofluid that is readily available, but which has not been extensively explored, is human milk, whose analysis could contribute to our understanding of the immune development of the newborn infant. Due to the occurrence of highly abundant lipids, proteins, and saccharides, milk peptidomics requires dedicated sample preparation steps. The aim of this study was to develop a time and cost-efficient workflow for the analysis of the human milk peptidome, for which we compared peptide extraction methodologies and peptide fragmentation methods. A method using strong acid protein precipitation and analysis by collision-induced dissociation fragmentation was found to be superior to all other test methods, allowing us qualitative and quantitative detection of about 4000 endogenous human milk peptides in a total analysis time of just 18 h.

Human milk peptides differentiate between the preterm and term infant and across varying lactational stages.

Variations in endogenous peptide profiles, functionality, and the enzymes responsible for the formation of these peptides in human milk are understudied. Additionally, there is a lack of knowledge regarding peptides in donor human milk, which is used to feed preterm infants when mother's own milk is not (sufficiently) available. To assess this, 29 human milk samples from the Dutch Human Milk Bank were analyzed as three groups, preterm late lactation stage (LS) (n = 12), term early (n = 8) and term late LS (n = 9). Gestational age (GA) groups were defined as preterm (24-36 weeks) and term (≥37 weeks). LS was determined as days postpartum as early (16-36 days) or late (55-88 days). Peptides, analyzed by LC-MS/MS, and parent proteins (proteins from matched peptide sequences) were identified and quantified, after which peptide functionality and the enzymes responsible for protein cleavage were determined. A total of 16 different parent proteins were identified from human milk, with no differences by GA or LS. We identified 1104 endogenous peptides, of which, the majority were from the parent proteins ß-casein, polymeric immunoglobulin receptor, αs1-casein, osteopontin, and κ-casein. The absolute number of peptides differed by GA and LS with 30 and 41 differing sequences respectively (p < 0.05) Odds likelihood tests determined that 32 peptides had a predicted bioactive functionality, with no significant differences between groups. Enzyme prediction analysis showed that plasmin/trypsin enzymes most likely cleaved the identified human milk peptides. These results explain some of the variation in endogenous peptides in human milk, leading to future targets that may be studied for functionality.

Lactational Stage of Pasteurized Human Donor Milk Contributes to Nutrient Limitations for Infants.

BACKGROUND: Mother's own milk is the first choice for feeding preterm infants, but when not available, pasteurized human donor milk (PDM) is often used. Infants fed PDM have difficulties maintaining appropriate growth velocities. To assess the most basic elements of nutrition, we tested the hypotheses that fatty acid and amino acid composition of PDM is highly variable and standard pooling practices attenuate variability; however, total nutrients may be limiting without supplementation due to late lactational stage of the milk. METHODS: A prospective cross-sectional sampling of milk was obtained from five donor milk banks located in Ohio, Michigan, Colorado, Texas-Ft Worth, and California. Milk samples were collected after Institutional Review Board (#07-0035) approval and informed consent. Fatty acid and amino acid contents were measured in milk from individual donors and donor pools (pooled per Human Milk Banking Association of North America guidelines). Statistical comparisons were performed using Kruskal-Wallis, Spearman's, or Multivariate Regression analyses with center as the fixed factor and lactational stage as co-variate. RESULTS: Ten of the fourteen fatty acids and seventeen of the nineteen amino acids analyzed differed across Banks in the individual milk samples. Pooling minimized these differences in amino acid and fatty acid contents. Concentrations of lysine and docosahexaenoic acid (DHA) were not different across Banks, but concentrations were low compared to recommended levels. CONCLUSIONS: Individual donor milk fatty acid and amino acid contents are highly variable. Standardized pooling practice reduces this variability. Lysine and DHA concentrations were consistently low across geographic regions in North America due to lactational stage of the milk, and thus not adequately addressed by pooling. Targeted supplementation is needed to optimize PDM, especially for the preterm or volume restricted infant.

Branched-chain fatty acid composition of human milk and the impact of maternal diet: the Global Exploration of Human Milk (GEHM) Study.

BACKGROUND: An understudied component of the diet, branched-chain fatty acids (BCFAs) are distinctive saturated fatty acids that may have an important influence on health. Human-milk fatty acid composition is known to differ worldwide, but comparative data are lacking on BCFAs. OBJECTIVE: We tested the hypotheses that concentrations of BCFAs in human milk differ between populations and are associated with maternal diet. DESIGN: We surveyed the BCFA composition of samples collected as part of a standardized, prospective study of human-milk composition. Mothers were enrolled from 3 urban populations with differing diets: Cincinnati, Ohio; Shanghai, China; and Mexico City, Mexico. Enrollment was limited to healthy mothers of term singleton infants. We undertook a cross-sectional analysis of milk from all women with samples at postpartum week 4 (n = 359; ∼120 women/site). Fatty acids were extracted from milk by using a modified Bligh-Dyer technique and analyzed by gas chromatography. Statistical analysis was performed by ANOVA and Tobit regression. For Cincinnati mothers, 24-h diet recalls were analyzed in relation to the individual BCFA concentrations measured in milk samples. RESULTS: Total BCFAs in milk differed by site, with the highest concentration in Cincinnati followed by Mexico City and Shanghai (mean ± SE: 7.90 ± 0.41, 6.10 ± 0.36, and 4.27 ± 0.25 mg/100 mL, respectively; P < 0.001). Site differences persisted after delivery mode, maternal age, and body mass index were controlled for. The individual concentrations of iso-14:0, iso-16:0, iso-18:0, anteiso-15:0, and anteiso-17:0 also differed between sites. Milk concentrations of iso-14:0 and anteiso-15:0 were associated with maternal intake of dairy; iso-16:0 was associated with maternal intakes of dairy and beef. CONCLUSIONS: BCFA concentrations in milk at 4 wk postpartum differed between mothers from Cincinnati, Shanghai, and Mexico City. Variations in human-milk BCFAs are influenced by diet. The impact of BCFAs on infant health warrants investigation.