Interlaboratory Comparison of Antibody-Free LC-MS/MS Measurements of C-peptide and Insulin.

BACKGROUND: The enhanced precision and selectivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it an attractive alternative to certain clinical immunoassays. Easily transferrable work flows could help facilitate harmonization and ensure high-quality patient care. We aimed to evaluate the interlaboratory comparability of antibody-free multiplexed insulin and C-peptide LC-MS/MS measurements. METHODS: The laboratories that comprise the Targeted Mass Spectrometry Assays for Diabetes and Obesity Research (TaMADOR) consortium verified the performance of a validated peptide-based assay (reproducibility, linearity, and lower limit of the measuring interval [LLMI]). An interlaboratory comparison study was then performed using shared calibrators, de-identified leftover laboratory samples, and reference materials. RESULTS: During verification, the measurements were precise (2.7% to 3.7%CV), linear (4 to 15 ng/mL for C-peptide and 2 to 14 ng/mL for insulin), and sensitive (LLMI of 0.04 to 0.10 ng/mL for C-peptide and 0.03 ng/mL for insulin). Median imprecision across the 3 laboratories was 13.4% (inter-quartile range [IQR] 11.6%) for C-peptide and 22.2% (IQR 20.9%) for insulin using individual measurements, and 10.8% (IQR 8.7%) and 15.3% (IQR 14.9%) for C-peptide and insulin, respectively, when replicate measurements were averaged. Method comparison with the University of Missouri reference method for C-peptide demonstrated a robust linear correlation with a slope of 1.044 and r2 = 0.99. CONCLUSIONS: Our results suggest that combined LC-MS/MS measurements of C-peptide and insulin are robust and adaptable and that standardization with a reference measurement procedure could allow accurate and precise measurements across sites, which could be important to diabetes research and help patient care in the future.

High Doses of Exogenous Glucagon Stimulate Insulin Secretion and Reduce Insulin Clearance in Healthy Humans.

Glucagon is generally defined as a counterregulatory hormone with a primary role to raise blood glucose concentrations by increasing endogenous glucose production (EGP) in response to hypoglycemia. However, glucagon has long been known to stimulate insulin release, and recent preclinical findings have supported a paracrine action of glucagon directly on islet ß-cells that augments their secretion. In mice, the insulinotropic effect of glucagon is glucose dependent and not present during basal euglycemia. To test the hypothesis that the relative effects of glucagon on hepatic and islet function also vary with blood glucose, a group of healthy subjects received glucagon (100 ng/kg) during fasting glycemia or experimental hyperglycemia (∼150 mg/dL) on 2 separate days. During fasting euglycemia, administration of glucagon caused blood glucose to rise due to increased EGP, with a delayed increase of insulin secretion. When given during experimental hyperglycemia, glucagon caused a rapid, threefold increase in insulin secretion, as well as a more gradual increase in EGP. Under both conditions, insulin clearance was decreased in response to glucagon infusion. The insulinotropic action of glucagon, which is proportional to the degree of blood glucose elevation, suggests distinct physiologic roles in the fasting and prandial states.

Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies.

Introduction: The measurement of insulin and C-peptide provides a valuable tool for the clinical evaluation of hypoglycemia. In research, these biomarkers are used together to better understand hyperinsulinemia, hepatic insulin clearance, and beta cell function. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an attractive approach for the analysis of insulin and C-peptide because the platform is specific, can avoid certain limitations of immunoassays, and can be multiplexed. Previously described LC-MS/MS methods for the simultaneous quantification of insulin and C-peptide measure the intact analytes and most have relied on immunoaffinity enrichment. These approaches can be limited in terms of sensitivity and interference from auto-antibodies, respectively. We have developed a novel method that does not require antibodies and uses proteolytic digestion to yield readily ionizable proteotypic peptides that enables the sensitive, specific, and simultaneous quantitation of insulin and C-peptide. Methods: Serum samples were precipitated with acetonitrile. Analytes were enriched using solid phase extraction and then digested with endoproteinase Glu-C. Surrogate peptides for insulin and C-peptide were analyzed using targeted LC-MS/MS. Results: Inter-day imprecision was below 20 %CV and linearity was observed down to the lower limit of quantitation for both analytes (insulin = 0.09 ng/mL, C-peptide = 0.06 ng/mL). Comparison to a commercially available insulin immunoassay (Beckman Coulter UniCel DxI 600 Access) revealed a 30% bias between methods. Conclusion: A novel LC-MS/MS method for the simultaneous analysis of insulin and C-peptide using Glu-C digestion was developed and evaluated. A detailed standard operating procedure is provided to help facilitate implementation in other laboratories.

Fucosylated Human Milk Oligosaccharide Foraging within the Species Bifidobacterium pseudocatenulatum Is Driven by Glycosyl Hydrolase Content and Specificity.

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.

Associations of Human Milk Oligosaccharides and Bioactive Proteins with Infant Morbidity and Inflammation in Malawian Mother-Infant Dyads.

BACKGROUND: Human milk oligosaccharides (HMOs) and bioactive proteins likely benefit infant health, but information on these relations is sparse. OBJECTIVES: We 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. METHODS: Breast milk samples at 6 mo postpartum (n  = 659) 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 12 mo, and CRP and AGP at 6 and 18 mo, 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. RESULTS: In secretors there were positive associations between total HMOs and longitudinal prevalence of fever (P = 0.032), between fucosylated HMOs and incidence of diarrhea (P = 0.026), and between lactoferrin and elevated CRP at 18 mo (P = 0.011). In nonsecretors, there were inverse associations between lactoferrin and incidence of fever (P  = 0.007), between osteopontin and longitudinal prevalence of lost appetite (P  = 0.038), and between fucosylated HMOs and incidence of diarrhea (P = 0.025), lost appetite (P = 0.019), and concentrations of AGP and CRP at 6 mo (P = 0.001 and 0.010); and positive associations between total HMOs and incidence of lost appetite (P = 0.024) and elevated CRP at 18 mo (P  = 0.026), between lactalbumin and incidence of diarrhea (P = 0.006), and between lactoferrin and elevated CRP at 18 mo (P = 0.015). CONCLUSIONS: Certain 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.

Associations of human milk oligosaccharides and bioactive proteins with infant growth and development among Malawian mother-infant dyads.

BACKGROUND: Human 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. OBJECTIVES: We 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. METHODS: Breast milk samples were analyzed at 6 mo (n = 659) 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. RESULTS: Among all participants, there were inverse associations of IgA and lactoferrin concentrations with motor skills (P = 0.018 and P = 0.044), and a positive association of lactalbumin concentration with motor skills (P = 0.038). Among secretors only [fucosyltransferase 2 gene (FUT2) positive], there were positive associations of absolute abundance of HMOs with ΔLAZ (P = 0.035), and relative abundance of fucosylated and sialylated HMOs with language at 18 mo (P < 0.001 and P = 0.033, respectively), and inverse associations of osteopontin with standing and walking at 12 mo (P = 0.007 and 0.002, respectively). Relative abundances of several individual HMOs were associated with growth and development, mostly among secretors. CONCLUSIONS: Certain 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.

High-throughput glycomic analyses reveal unique oligosaccharide profiles of canine and feline milk samples.

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<0.05) in oligosaccharide abundances were observed between collection time points and between diet groups within species. These data explore the oligosaccharide content of canine and feline maternal milk, representing an opportunity to generate a fundamental understanding of the nutritional needs of new-born puppies and kittens.

Deep Structural Analysis and Quantitation of O-Linked Glycans on Cell Membrane Reveal High Abundances and Distinct Glycomic Profiles Associated with Cell Type and Stages of Differentiation.

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.

Immunoglobulin A N-glycosylation Presents Important Body Fluid-specific Variations in Lactating Mothers.

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 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. Most of these unique N-Glycan compositions identified in SIgA from mature milk and IgA from plasma were fucosylated and both fucosylated and sialylated species, whereas 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.

Selective Proteolysis of α-Lactalbumin by Endogenous Enzymes of Human Milk at Acidic pH.

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 Proteins and Their Glycosylation Exhibit Quantitative Dynamic Variations during Lactation.

BACKGROUND: Proteins 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. OBJECTIVE: This 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. DESIGN: A 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. RESULTS: α-Lactalbumin, ß-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 < 0.002). α-Lactalbumin (37%), ß-casein (9%), and lysozyme (159%) were higher in mature milk than in colostrum. Glycans exhibited different behavior. Fucosylated glycans of lactoferrin and high-mannose, undecorated, fucosylated, sialylated, and combined fucosylated + sialylated glycans of secretory immunoglobulin A increased during lactation even when the concentrations of the parent proteins decreased. CONCLUSIONS: Proteins in healthy mothers vary dynamically through lactation to support the development of infants. Individual milk proteins carried unique glycan modifications that varied systematically in structure even with site specificity. The role of glycosylation in human milk proteins will be important in understanding the functional components of human milk. This trial was registered at clinicaltrials.gov as NCT01817127.

Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses.

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.

Composition and Variation of Macronutrients, Immune Proteins, and Human Milk Oligosaccharides in Human Milk From Nonprofit and Commercial Milk Banks.

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.

Lipid-Based Nutrient Supplements During Pregnancy and Lactation Did Not Affect Human Milk Oligosaccharides and Bioactive Proteins in a Randomized Trial.

BACKGROUND: Human milk oligosaccharides (HMOs) and bioactive proteins are beneficial to infant health. Recent evidence suggests that maternal nutrition may affect the amount of HMOs and proteins in breast milk; however, the effect of nutrient supplementation on HMOs and bioactive proteins has not yet been well studied. OBJECTIVE: We aimed to determine whether lipid-based nutrient supplements (LNSs) affect milk bioactive protein and HMO concentrations at 6 mo postpartum in women in rural Malawi. These are secondary outcomes of a previously published randomized controlled trial. METHODS: Women were randomly assigned to consume either an iron and folic acid capsule (IFA) daily from ≤20 wk gestation until delivery, followed by placebo daily from delivery to 6 mo postpartum, or a multiple micronutrient (MMN) capsule or LNS daily from ≤20 wk gestation to 6 mo postpartum. Breast milk concentrations of total HMOs, sialylated HMOs, fucosylated HMOs, lactoferrin, lactalbumin, lysozymes, antitrypsin, immunoglobulin A, and osteopontin were analyzed at 6 mo postpartum (n = 647). Between-group differences in concentrations and in proportions of women classified as having low concentrations were tested. RESULTS: HMO and bioactive protein concentrations did not differ between groups (P > 0.10 for all comparisons). At 6 mo postpartum, the proportions of women with low HMOs or bioactive proteins were not different between groups except for osteopontin. A lower proportion of women in the IFA group had low osteopontin compared with the LNS group after adjusting for covariates (OR: 0.5; 95% CI: 0.3, 0.9; P = 0.016). CONCLUSION: The study findings do not support the hypothesis that supplementation with an LNS or MMN capsule during pregnancy and postpartum would increase HMO or bioactive milk proteins at 6 mo postpartum among Malawian women. This trial was registered at clinicaltrials.gov as NCT01239693.

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM).

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.

Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation.

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.

Quantitation of Site-Specific Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs.

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 ultrahigh-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 multilevel structural characterization is important for the successful development of therapeutic antibodies.