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Proteins on cell membrane are modified by N- and O-glycans. N-Glycans have been extensively characterized using advanced separation and mass spectrometry techniques. However, O-glycans remain a challenge, because of the lack of universal enzymes to release them and the large background abundances of N-glycans. Here, we report a method for in-depth structural analysis and quantitation of O-glycans derived from human cell membrane. O-Glycans were chemically released from isolated cell membrane glycoproteins following N-glycan and lipid/glycolipid removal by PNGase F digestion and Folch extraction, respectively. Released O-glycans were purified by an optimized protocol to eliminate interference from small molecules and degraded proteins. Cell surface O-glycans were then analyzed using a nanoLC-chip-QTOF mass spectrometer with a porous graphitized carbon (PGC) column, while the N-glycans and glycolipids isolated from the same cell membrane fractions were analyzed in parallel using previously reported methods. The monosaccharide compositions and linkages of the detected O-glycans were identified by exoglycosidase digestion facilitated with tandem mass spectrometry (MS/MS). Using this method, we identified 44 cell membrane O-glycan isomers with MS/MS, and, among them, we unambiguously characterized 25 O-glycan structures with exoglycosidase digestion to create a library with their complete structures, accurate masses, and retention times. In this process, we identified and characterized unexpected mannose oligomers that are α(1-2/3) linked. This library enabled the identification and quantification of unique cell surface O-glycans from different cell lines and the study of specific O-glycan changes during cell differentiation.

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs), we observed two distinct groups of B. pseudocatenulatum, isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 α-fucosidases, consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO-binding specificities of the family 1 solute-binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain's distinct HMO consumption pattern. Taken together, these data indicate the presence or absence of specific α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ. IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breastfed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies key gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

Author(s): Jorgensen, Josh M; Young, Rebecca; Ashorn, Per; Ashorn, Ulla; Chaima, David; Davis, Jasmine CC; Goonatilleke, Elisha; Kumwenda, Chiza; Lebrilla, Carlito B; Maleta, Kenneth; Sadalaki, John; Totten, Sarah M; Wu, Lauren D; Zivkovic, Angela M; Dewey, Kathryn G | Abstract: BackgroundHuman milk oligosaccharides (HMOs) and bioactive proteins likely benefit infant health, but information on these relations is sparse.ObjectivesWe aimed to examine associations of milk content of HMOs and bioactive proteins with incidence and longitudinal prevalence of infant morbidity (any illness, fever, diarrhea, acute respiratory infection, and loss of appetite) and markers of inflammation [C-reactive protein (CRP) and α-1-acid glycoprotein (AGP)]. These are secondary analyses of a randomized controlled trial.MethodsBreast milk samples at 6nmo postpartum (nnn=n659) were analyzed to quantify absolute abundance of HMOs, relative abundance of fucosylated HMOs, sialylated HMOs, and 51 individual HMOs, and concentrations of 6 bioactive proteins (lactalbumin, lactoferrin, lysozyme, antitrypsin, IgA, and osteopontin). We examined associations of these constituents with infant morbidity from 6 to 7 and 6 to 12nmo, and CRP and AGP at 6 and 18nmo, considering maternal secretor status [presence or absence of the functional enzyme encoded by the fucosyltransferase 2 gene (FUT2) ] and adjusting for covariates and multiple hypothesis testing.ResultsIn secretors there were positive associations between total HMOs and longitudinal prevalence of fever (Pn=n0.032), between fucosylated HMOs and incidence of diarrhea (Pn=n0.026), and between lactoferrin and elevated CRP at 18nmo (Pn=n0.011). In nonsecretors, there were inverse associations between lactoferrin and incidence of fever (Pnn=n0.007), between osteopontin and longitudinal prevalence of lost appetite (Pnn=n0.038), and between fucosylated HMOs and incidence of diarrhea (Pn=n0.025), lost appetite (Pn=n0.019), and concentrations of AGP and CRP at 6nmo (Pn=n0.001 and 0.010); and positive associations between total HMOs and incidence of lost appetite (Pn=n0.024) and elevated CRP at 18nmo (Pnn=n0.026), between lactalbumin and incidence of diarrhea (Pn=n0.006), and between lactoferrin and elevated CRP at 18nmo (Pn=n0.015).ConclusionsCertain HMOs and bioactive proteins were associated with infant morbidity and inflammation, particularly in nonsecretors. Further research is needed to elucidate the causality of these relations.This trial was registered at clinicaltrials.gov as NCT01239693.

Author(s): Jorgensen, Josh M; Young, Rebecca; Ashorn, Per; Ashorn, Ulla; Chaima, David; Davis, Jasmine CC; Goonatilleke, Elisha; Kumwenda, Chiza; Lebrilla, Carlito B; Maleta, Kenneth; Prado, Elizabeth L; Sadalaki, John; Totten, Sarah M; Wu, Lauren D; Zivkovic, Angela M; Dewey, Kathryn G | Abstract: BackgroundHuman milk oligosaccharides (HMOs) and bioactive breast milk proteins have many beneficial properties. Information is sparse regarding associations between these milk constituents and infant growth and development in lower-income countries.ObjectivesWe aimed to examine associations of milk content of HMOs and bioactive proteins at 6 mo postpartum with infant growth and motor and cognitive development. These are secondary analyses of a randomized controlled trial in rural Malawi.MethodsBreast milk samples were analyzed at 6 mo (nn=n659) for general categories of HMOs (total HMOs, fucosylated HMOs, and sialylated HMOs), 51 individual HMOs, and 6 bioactive proteins (lactalbumin, lactoferrin, lysozyme, antitrypsin, IgA, and osteopontin). We examined associations of the relative abundances of HMOs and concentrations of bioactive proteins with infant growth from 6 to 12 mo [change in length-for-age (ΔLAZ), weight-for-age, weight-for-length, and head circumference z-scores] as well as ability to stand or walk alone at 12 mo, and motor and language skills, socioemotional development, executive function, and working memory at 18 mo. Analyses were adjusted for covariates and multiple hypothesis testing.ResultsAmong all participants, there were inverse associations of IgA and lactoferrin concentrations with motor skills (Pn=n0.018 and P =n0.044), and a positive association of lactalbumin concentration with motor skills (Pn=n0.038). Among secretors only [fucosyltransferase 2 gene (FUT2) positive], there were positive associations of absolute abundance of HMOs with ΔLAZ (Pn=n0.035), and relative abundance of fucosylated and sialylated HMOs with language at 18 mo (Pnln0.001 and Pn=n0.033, respectively), and inverse associations of osteopontin with standing and walking at 12 mo (Pn=n0.007 and 0.002, respectively). Relative abundances of several individual HMOs were associated with growth and development, mostly among secretors.ConclusionsCertain bioactive breast milk proteins and HMOs are associated with infant growth and motor and cognitive development. Further studies are needed to determine if a causal relation exists.This trial was registered at clinicaltrials.gov as NCT01239693.

Background Tears play an important role in ocular surface protection, and help wild animals maintain visual acuity in the face of air and water friction. The proteomics of tears has only been described for mammals. The knowledge of the proteomics of wild animal tears can aid not only in the setting of normal standards for ocular disease studies in these animals, but also to base the search for new molecules to be used in ophthalmology therapeutics. We therefore set out to describe the proteomic profile of roadside hawk (Rupornis magnirostris), broad-snouted caiman (Caiman latirostris) and loggerhead sea turtle (Caretta caretta) tears. Tears were collected from healthy animals, their spectral profiles were obtained with an LTQ Orbitrap XL mass spectrometer, and the dataset was analyzed against reference taxa. Results For roadside hawk, 446 proteins were identified, the most abundant being albumin, transferrin, globulin and actin. For broad-snouted caiman and loggerhead sea turtle, 1358 and 163 proteins were identified, respectively. Uncharacterized proteins and transferrin were highly abundant in both species. The roadside hawk tear components and their properties were similar to those described for humans, but with a higher albumin concentration. Broad-snouted caiman tears presented a wide diversity of ontological functions, with an abundant presence of enzymatic compounds. In loggerhead sea turtle tears, the predominance of proteins with ion-transport functions was consistent with possible osmolality-maintenance mechanisms. Conclusion These data enhance our understanding of birds and reptiles’ tears microcomposition and may be used to base the discovery of new molecules with high biotechnological potential.

Oligosaccharides are important components of milk, serving as substrates for the intestinal microbiota, acting as antimicrobials that prevent pathogen colonization, and supporting the developing gastrointestinal immune system of neonates. Nutrient composition of canine and feline milk samples has been described previously, but little is known about the oligosaccharide content. Therefore, the objective of this study was to characterize canine and feline milk samples using a high-throughput glycomics approach. 23 dogs (9 Labrador retriever and 14 Labrador retriever x golden retriever crossbreed) and 6 domestic shorthair cats were recruited to the study. Milk samples were collected by manual expression at time points after parturition. Samples were collected across 2 phases per species, differentiated by maternal diet. Following extraction, oligosaccharide content was determined by liquid chromatography-mass spectrometry (LC-MS). In canine milk samples, 3 structures accounted for over 90% of all oligosaccharides detected across two diet groups. These were 3’-sialyllactose, 6’-sialyllactose, and 2’-fucosyllactose. In feline samples, a more diverse range of oligosaccharides was detected, with up to 16 structures present at relative abundance >1% of the total. Difucosyllactose-N-hexaose b, 3’-sialyllactose and lacto-N-neohexaose were all detected at abundances >10% in feline milk samples. Statistically significant differences (p

The review focuses on recent aspects (last three years) of glycosylation analyses that provide relevant information about cancer. It includes recent development in glycan and protein enrichment methods for discovery of cancer markers. It will however focus on the recent technological developments in mass spectrometry (MS), bioinformatics and separation methods as they apply toward identifying cancer markers. More specifically, it will cover advances in matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), capillary electrophoresis (CE), and liquid chromatography (LC) coupled to mass spectrometry. The discussions will include glycans, recently identified as potential markers for cancer that have been discovered using the highlighted technologies. We will also discuss emerging glycoproteomic techniques and site-specific methods, and how these methods are being utilized for cancer biomarker discovery. The large amount of data and the complexity of glycoproteomic analysis have been the impetus for developing bioinformatic methods for assigning glycosylation sites and characterizing the potentially very large site- or microheterogeneity. This review will cover the most recent advancements in biomarker discovery of N- and O-glycosylation of proteins as well as the glycolipids. This group collectively constitutes glycosylation on the cell membrane or the glycocalyx. The review will also highlight methods that are highly reproducible, with low coefficient of variation (CV), and scalable for large sample sets. The reader is also referred to other notable earlier reviews on glycomic biomarkers for cancer. Mereiter et al. describe the recent glycomic effort in gastrointestinal cancer.1 A review focused on N-glycomic analysis of colorectal cancer has been published by Sethi and Fanayan.2 N-Glycan, O-glycan, and glycolipid characteristics of colorectal cancer were reviewed by Holst et al.3 Muchena et al. have provided a more general review of glycan biomarkers covering up to the current review period.4 The field of glycoscience also covers a broad area of structures and may include highly anionic (glycosaminoglycans) and monosaccharide (e.g. O-GlcNAc) modifications that require their specific and unique sets of analytical tools. The latter topics are not covered in this review. 1.1. Background of Glycosylation and Cancer There is nearly 50 years of research illustrating that changes in glycosylation accompany cancer.5 Glycosylation is a dynamic process intimately involved in key processes in cells, including cell-cell and cell-extracellular communication as well as cell-cell adhesion, and cellular metabolism. Glycans expressed in several types of glycoconjugates are known to change during cancer genesis and progression.6 These changes increase the structural heterogeneity and alter the functions of cells.7 Glycosylation has been found to enable tumor-induced immunomodulation and metastasis.8–10 The cell-surface structures allow the immune cells to differentiate self/normal cells from non-self/abnormal cells.11 For example, terminal residues on N-glycans, such as sialic acids, are involved in immunity and cell-cell communication.12 Changes in glycosylation of adhesion proteins can largely influence their binding properties, leading to altered cell-cell or cell-matrix contacts.13 Other types of glycans are also involved in cancer. Gangliosides and sphingolipids are involved in transmembrane communication vital in tumor cell growth and invasion.14 Glycosaminoglycans are involved in tumor cell migration15 and motility.16–18 The search for effective markers is aided by the understanding of how glycans are synthesized. The glycan biosynthetic process is a non-template process involving multiple enzymes, some performing competing activities. It is estimated that more than 300 metabolic enzymes, composed of glycosyltransferases and glycosidases, are involved in the biosynthesis and processing of glycans.19–20 The best-known series of pathways belongs to the production of N-glycans (Figure 1). They illustrate the large degree of structural heterogeneity in glycosylation. N-Glycans are produced in a step-wise process beginning with the production of high mannose structures on a lipid, which are transferred to the nascent polypeptide chain to guide protein folding. Once folded, the glycans are then trimmed back and extended to form complex and hybrid structures. The folded protein can be secreted with glycans that range from early in the process to yield high mannose structures to later in the process corresponding to complex or hybrid structures. The number of structures for one glycosylation site can vary by a large degree, from a handful for transferrin21 to over 70 structures for IgG, the most abundant serum glycoprotein.22–23 Open in a separate window Figure 1. Representation of the glycosylation pathway of proteins. The pathway illustrates the complexity and heterogeneity of structures. The proteins may exit the pathway with various levels of glycosylation.

BackgroundProteins in human milk are essential and known to support the growth, development, protection, and health of the newborn. These proteins are highly modified by glycans that are currently being recognized as vital to protein structure, stability, function, and health of the intestinal mucosa. Although milk proteins have been studied, the quantitative changes in milk proteins and their respective site-specific glycosylation are unknown.ObjectiveThis study expanded the analytical tools for milk proteins and their site-specific glycosylation and applied these tools to a large cohort to determine changes in individual protein concentrations and their site-specific N-glycosylation across lactation.DesignA tandem mass spectrometry method was applied to 231 breast-milk samples from 33 mothers in Davis, California, obtained during 7 different periods of lactation. Dynamic changes in the absolute abundances of milk proteins, as well as variation in site-specific N-glycosylation of individual proteins, were quantified.Resultsα-Lactalbumin, β-casein, k-casein, and α-antitrypsin were significantly increased from colostrum to transitional milk (4.37±1.33 g/L to 6.41±0.72 g/L, 2.25±0.86 g/L to 2.59±0.78 g/L, 1.33±0.44 g/L to 1.60±0.39 g/L, and 0.09±0.10 g/L to 0.11±0.04 g/L, respectively; P&nbsp

Secretory Immunoglobulin A (SIgA) is central to mucosal immunity: represents one of the main immunological mechanisms of defense against the potential attack of pathogens. During lactation SIgA is produced by plasmablasts in the mammary gland and is present in breast milk, playing a vital role in the passive immunity of the newborn. Interestingly, the different components of SIgA are highly N-glycosylated, and these N-Glycans have an essential role in health maintenance. In this work, we performed a glycomic study to compare N-glycosylation of SIgA purified from mature breast milk and saliva, and also plasma IgA from the same lactating participants. Our results revealed a greater diversity than previously reported, with 89 glycan compositions that may correspond to over 250 structures. Among these glycans, 54 glycan compositions were characterized as body-fluid specific. The majority of these unique N-Glycan compositions identified in SIgA from mature milk and IgA from plasma were fucosylated and both fucosylated and sialylated species, while in salivary SIgA the unique structures were mainly undecorated complex N-Glycans. In addition, we evaluated the effect of delivery mode on (S)IgA glycosylation. Lactating participants who had given birth by vaginal delivery presented an increased proportion of high mannose and fucosylated glycans in salivary SIgA, and selected high mannose, fucosylated, sialylated, and both fucosylated and sialylated glycans in plasma IgA, indicating that the hormonal changes during vaginal delivery could affect plasma and saliva IgA. These results reveal the structural details that provide a new dimension to the roles of (S)IgA N-Glycans in different tissues, and especially in maternal and new-born protection and infant development. The design of optimal recombinant IgA molecules specifically targeted to protect mucosal surfaces will need to include this dimension of structural detail. Fil: Goonatilleke, Elisha. University of California at Davis; Estados Unidos Fil: Smilowitz, Jennifer T.. University of California at Davis; Estados Unidos Fil: Mariño, Karina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: German, Bruce J.. University of California at Davis; Estados Unidos Fil: Lebrilla, Carlito. University of California at Davis; Estados Unidos Fil: Barboza, Mariana. University of California at Davis; Estados Unidos

During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultra high performance liquid chromatography – triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multi-level structural characterization is important for the successful development of therapeutic antibodies.

Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycans are produced through a complicated nontemplate driven process involving the competition of enzymes that extend the nascent chain. The large diversity of structures, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies of glycans all conspire to make the analysis arguably much more difficult than any other biopolymer. Furthermore, the large number of glycoforms associated with a specific protein site makes it more difficult to characterize than any post-translational modification. Nonetheless, there have been significant progress, and advanced separation and mass spectrometry methods have been at its center and the main reason for the progress. While glycomic and glycoproteomic analyses are still typically available only through highly specialized laboratories, new software and workflow is making it more accessible. This review focuses on the role of mass spectrometry and separation methods in advancing glycomic and glycoproteomic analyses. It describes the current state of the field and progress toward making it more available to the larger scientific community.

Background:When human milk is unavailable, banked milk is recommended for feeding premature infants. Milk banks use processes to eliminate pathogens; however, variability among methods exists.Research aim:The aim of this study was to compare the macronutrient (protein, carbohydrate, fat, energy), immune-protective protein, and human milk oligosaccharide (HMO) content of human milk from three independent milk banks that use pasteurization (Holder vs. vat techniques) or retort sterilization.Methods:Randomly acquired human milk samples from three different milk banks ( n = 3 from each bank) were analyzed for macronutrient concentrations using a Fourier transform mid-infrared spectroscopy human milk analyzer. The concentrations of IgA, IgM, IgG, lactoferrin, lysozyme, α-lactalbumin, α antitrypsin, casein, and HMO were analyzed by mass spectrometry.Results:The concentrations of protein and fat were significantly ( p < .05) less in the retort sterilized compared with the Holder and vat pasteurized samples, respectively. The concentrations of all immune-modulating proteins were significantly ( p < .05) less in the retort sterilized samples compared with vat and/or Holder pasteurized samples. The total HMO concentration and HMOs containing fucose, sialic acid, and nonfucosylated neutral sugars were significantly ( p < .05) less in retort sterilized compared with Holder pasteurized samples.Conclusion:Random milk samples that had undergone retort sterilization had significantly less immune-protective proteins and total and specific HMOs compared with samples that had undergone Holder and vat pasteurization. These data suggest that further analysis of the effect of retort sterilization on human milk components is needed prior to widespread adoption of this process.

Scope The use of human milk products is increasing for high-risk infants. Human milk contains endogenous enzymes that comprise a dynamic proteolytic system, yet biological properties of these enzymes and their activities in response to variations including pH within infants are unclear. Human milk has a neutral pH around 7, while infant gastric pH varies from 2 to 6 depending on individual conditions. This study is designed to determine the specificity of enzyme-substrate interactions in human milk as a function of pH. Methods and results Endogenous proteolysis is characterized by incubating freshly expressed human milk at physiologically relevant pH ranging from 2 to 7 without the addition of exogenous enzymes. Results show that the effects of pH on endogenous proteolysis in human milk are protein-specific. Further, specific interactions between cathepsin D and α-lactalbumin are confirmed. The endogenous enzyme cathepsin D in human milk cleaves α-lactalbumin as the milk pH shifts from 7 to 3. Conclusions This study documents that selective proteolysis activated by pH shift is a mechanism for dynamic interactions between human milk and the infant. Controlled proteolysis can guide the use of human milk products based on individual circumstance.

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.

BACKGROUND The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards. OBJECTIVE The objective of our study was to rapidly measure the absolute concentrations of HMOs in milk using LC-mass spectrometry (MS) and to determine the phenotypic secretor status of the mothers. METHODS This quantitative method for measuring HMO concentration was developed by using ultraperformance LC multiple reaction monitoring MS. It was validated and applied to milk samples from Malawi (88 individuals; 88 samples from postnatal month 6) and the United States (Davis, California; 45 individuals, mean age: 32 y; 103 samples collected on postnatal days 10, 26, 71, or 120, repeated measures included). The concentrations of α(1,2)-fucosylated HMOs were used to determine the mothers' phenotypic secretor status with high sensitivity and specificity. We used Friedman's test and Wilcoxon's signed rank test to evaluate the change in HMO concentration during the course of lactation, and Student's t test was used to compare secretors and nonsecretors. RESULTS A decrease (P < 0.05) in HMO concentration was observed during the course of lactation for the US mothers, corresponding to 19.3 ± 2.9 g/L for milk collected on postnatal day 10, decreasing to 8.53 ± 1.18 g/L on day 120 (repeated measures; n = 14). On postnatal day 180, the total concentration of HMOs in Malawi milk samples from secretors (6.46 ± 1.74 mg/mL) was higher (P < 0.05) than that in samples from nonsecretors (5.25 ± 2.55 mg/mL ). The same trend was observed for fucosylated species; the concentration was higher in Malawi milk samples from secretors (4.91 ± 1.22 mg/mL) than from nonsecretors (3.42 ± 2.27 mg/mL) (P < 0.05). CONCLUSIONS HMOs significantly decrease during the course of lactation. Secretor milk contains higher concentrations of total and fucosylated HMOs than does nonsecretor milk. These HMO concentrations can be correlated to the health of breastfed infants in order to investigate the protective effects of milk components. The trials were registered at clinicaltrials.gov as NCT01817127 and NCT00524446.