Nutritional and Clinical Glycomics Research

Author: laurenmd (Page 1 of 9)

New Publication in Cell!

Sialylated Milk Oligosaccharides Promote Microbiota-Dependent Growth in Models of Infant Undernutrition

Mark R. Charbonneau, David O’Donnell, Laura V. Blanton, Sarah M. Totten, Jasmine C.C. Davis, Michael J. Barratt, Jiye Cheng, Janaki Guruge, Michael Talcott, James R. Bain, Michael J. Muehlbauer, Olga Ilkayeva, Chao Wu, Tedd Struckmeyer, Daniela Barile, Charles Mangani, Josh Jorgensen, Yue-mei Fan, Kenneth Maleta, Kathryn G. Dewey, Per Ashorn, Christopher B. Newgard, Carlito Lebrilla, David A. Mills, Jeffrey I. Gordon

Identifying interventions that more effectively promote healthy growth of children with undernutrition is a pressing global health goal. Analysis of human milk oligosaccharides (HMOs) from 6-month-postpartum mothers in two Malawian birth cohorts revealed that sialylated HMOs are significantly less abundant in those with severely stunted infants. To explore this association, we colonized young germ-free mice with a consortium of bacterial strains cultured from the fecal microbiota of a 6-month-old stunted Malawian infant and fed recipient animals a prototypic Malawian diet with or without purified sialylated bovine milk oligosaccharides (S-BMO). S-BMO produced a microbiota-dependent augmentation of lean body mass gain, changed bone morphology, and altered liver, muscle, and brain metabolism in ways indicative of a greater ability to utilize nutrients for anabolism. These effects were also documented in gnotobiotic piglets using the same consortium and Malawian diet. These preclinical models indicate a causal, microbiota-dependent relationship between S-BMO and growth promotion.

New Blog Post!

After a year of working in the Lebrilla lab on disease biomarkers, Nan was ready to head back to her university in Shanghai.  We had a farewell party at Taste of Thai in Davis.  She’s made much progress in her field of research during her stay, and also was able to make good memories with the group.  Matthew Amicucci was able to sit down and interview Nan about her time here.   Click here and get to know Nan Yang.

 

New Publication!

The impact of freeze-drying infant fecal samples on measures of their bacterial community profiles and milk-derived oligosaccharide content

1,2, 2,3, 1,2, 1,2,2,3, 1,2,4

Abstract:

Infant fecal samples are commonly studied to investigate the impacts of breastfeeding on the development of the microbiota and subsequent health effects. Comparisons of infants living in different geographic regions and environmental contexts are needed to aid our understanding of evolutionarily-selected milk adaptations. However, the preservation of fecal samples from individuals in remote locales until they can be processed can be a challenge. Freeze-drying (lyophilization) offers a cost-effective way to preserve some biological samples for transport and analysis at a later date. Currently, it is unknown what, if any, biases are introduced into various analyses by the freeze-drying process. Here, we investigated how freeze-drying affected analysis of two relevant and intertwined aspects of infant fecal samples, marker gene amplicon sequencing of the bacterial community and the fecal oligosaccharide profile (undigested human milk oligosaccharides). No differences were discovered between the fecal oligosaccharide profiles of wet and freeze-dried samples. The marker gene sequencing data showed an increase in proportional representation ofBacteriodes and a decrease in detection of bifidobacteria and members of class Bacilli after freeze-drying. This sample treatment bias may possibly be related to the cell morphology of these different taxa (Gram status). However, these effects did not overwhelm the natural variation among individuals, as the community data still strongly grouped by subject and not by freeze-drying status. We also found that compensating for sample concentration during freeze-drying, while not necessary, was also not detrimental. Freeze-drying may therefore be an acceptable method of sample preservation and mass reduction for some studies of microbial ecology and milk glycan analysis.

2015 Publications

Dallas, D.C., A. Guerrero, E.A. Parker, R.C. Robinson, J. Gan, J.B. German, D. Barile, and C.B. Lebrilla, Current peptidomics- Applications, purification, identification, quantification, and functional analysis. Proteomics, 2015. 15(5-6): p. 1026-38.

Dallas, D.C., C.J. Smink, R.C. Robinson, T. Tian, A. Guerrero, E.A. Parker, J.T. Smilowitz, K.A. Hettinga, M.A. Underwood, C.B. Lebrilla, J.B. German, and D. Barile, Endogenous Human Milk Peptide Release Is Greater after Preterm Birth than Term Birth. J Nutr, 2015. 145(3): p. 425-33.

De Leoz, M.L., K.M. Kalanetra, N.A. Bokulich, J.S. Strum, M.A. Underwood, J.B. German, D.A. Mills, and C.B. Lebrilla, Human Milk Glycomics and Gut Microbial Genomics in Infant Feces Show a Correlation between Human Milk Oligosaccharides and Gut Microbiota- A Proof-of-Concept Study. J Proteome Res, 2015. 14(1): p. 491-502.

Guerrero, A., D.C. Dallas, S. Contreras, A. Bhandari, A. Canovas, A. Islas-Trejo, J.F. Medrano, E.A. Parker, M. Wang, K. Hettinga, S. Chee, J.B. German, D. Barile, and C.B. Lebrilla, Peptidomic analysis of healthy and subclinically mastitic bovine milk. Int Dairy J, 2015. 46: p. 46-52.

Guerrero, A., L. Lerno, D. Barile, and C.B. Lebrilla, Top-Down Analysis of Highly Post-Translationally Modified Peptides by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J Am Soc Mass Spectrom, 2015. 26(3): p. 453-9.

Hong, Q., L.R. Ruhaak, C. Stroble, E. Parker, J. Huang, E. Maverakis, and C.B. Lebrilla, A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins. J Proteome Res, 2015.

Huang, J., A. Guerrero, E. Parker, J.S. Strum, J.T. Smilowitz, J.B. German, and C.B. Lebrilla, Site-specific Glycosylation of Secretory Immunoglobulin A from Human Colostrum.  J Proteome Res, 2015. 14(3): p. 1335-49.

Krishnan, S., J. Huang, H. Lee, A. Guerrero, L. Berglund, A. Erdembileg, C.B. Lebrilla, and A.M. Zivkovic, Combined HDL proteomic and glycomic profiles in patients at risk for coronary artery disease.. J Proteome Res, 2015.

Lewis, Z.T., S.M. Totten, J.T. Smilowitz, M. Popovic, E. Parker, D.G. Lemay, M.L. Van Tassell, M.J. Miller, Y.S. Jin, J.B. German, C.B. Lebrilla, and D.A. Mills, Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants.. Microbiome, 2015. 3: p. 13.

Maverakis, E., K. Kim, M. Shimoda, M.E. Gershwin, F. Patel, R. Wilken, S. Raychaudhuri, L.R. Ruhaak, and C.B. Lebrilla, Glycans in the immune system and The Altered Glycan Theory of Autoimmunity- A critical review. J Autoimmun, 2015. 57: p. 1-13.
Mechref, Y. and C. Lebrilla, 30th ASMS Asilomar Conference on Advances in Glycomics and Glycoproteomics: Methods and Applications. J Am Soc Mass Spectrom, 2015. 26(7): p. 1047-50.

Park, D., K.A. Brune, A. Mitra, A.I. Marusina, E. Maverakis, and C.B. Lebrilla, Characteristic changes in cell surface glycosylation accompany intestinal epithelial cell differentiation: high mannose structures dominate the cell surface glycome of undifferentiated enterocytes. Mol Cell Proteomics, 2015.

Ruhaak, L.R., D.A. Barkauskas, J. Torres, C.L. Cooke, L.D. Wu, C. Stroble, S. Ozcan, C.C. Williams, M. Camorlinga, D.M. Rocke, C.B. Lebrilla, and J.V. Solnick,The serum immunoglobulin G glycosylation signature of gastric cancer EuPA Open Proteom, 2015. 6: p. 1-9.

Ruhaak, L.R. and C.B. Lebrilla, Applications of Multiple Reaction Monitoring to Clinical Glycomics, 2015. 78(5-6): p. 335-342.

Ruhaak, L.R., S.L. Taylor, C. Stroble, U.T. Nguyen, E.A. Parker, T. Song, C.B. Lebrilla, W.N. Rom, H. Pass, K. Kim, K. Kelly, and S. Miyamoto, Differential N-glycosylation patterns in lung adenocarcinoma tissue. J Proteome Res, 2015.

Song, T., D.L. Aldredge, and C.B. Lebrilla, A method for in-depth structural annotation of human serum glycans yields the biological variations. Anal Chem, 2015.

Underwood, M.A., J.B. German, C.B. Lebrilla, and D.A. Mills, Bifidobacterium longum subspecies infantis: champion colonizer of the infant gut. Pediatr Res, 2015. 77(1-2): p. 229-35.

Wang, M., M. Li, S. Wu, C.B. Lebrilla, R.S. Chapkin, I. Ivanov, and S.M. Donovan, Fecal microbiota composition of breast-fed infants is correlated with human milk oligosaccharides consumed.. J Pediatr Gastroenterol Nutr, 2015. 60(6): p. 825-33.

New Website

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New Publications!

Hong, Q., et al. (2015). “A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.” J Proteome Res.
A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and non-specific enzyme digestions followed by nano LC-Chip/QTOF mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20-min UPLC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

Krishnan, S., et al. (2015). “Combined HDL proteomic and glycomic profiles in patients at risk for coronary artery disease..” J Proteome Res.
OBJECTIVES: To test whether recently developed methods for comprehensive profiling of the HDL glycome combined with the HDL proteome can distinguish individuals with coronary artery disease (CAD) from those without. METHODS: Twenty subjects, at risk for CAD, who underwent diagnostic coronary arteriography were analyzed. Ten subjects had CAD, and ten did not. HDL were extracted from fasting plasma samples by ultracentrifugation, followed by shotgun proteomic, glycomic and ganglioside analyses using LC-MS. CAD vs. non-CAD subjects’ data were compared using univariate and multivariate statistics. RESULTS: Principal components analysis showed a clear separation of CAD and non-CAD subjects, confirming that combined HDL proteomic and glycomic profiles distinguished at-risk subjects with atherosclerosis from those without. CAD patients had lower HDL apolipoprotein content (specifically Apo AI, AII and E, p <0.05), and lower serum amyloid A2 (SAA2, p = 0.020) and SAA4 (p = 0.007) but higher sialylated glycans (p<0.05). CONCLUSION: Combined proteomic and glycomic profiling of isolated HDL was tested as a novel analytical approach for developing biomarkers of disease. In this pilot study we found that HDL proteome and glycome distinguished between individuals who had CAD from those who did not within a group of individuals equally at risk for heart disease.

Park, D., et al. (2015). “Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes.” Mol Cell Proteomics.
Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Due to their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract.

 

New Publications!

Dallas, D. C., et al. (2014). “Current peptidomics- Applications, purification, identification, quantification, and functional analysis” Proteomics.
Peptidomics is an emerging field branching from proteomics that targets endogenously produced protein fragments. Endogenous peptides are often functional within the body-and can be both beneficial and detrimental. This review covers the use of peptidomics in understanding digestion, and identifying functional peptides and biomarkers. Various techniques for peptide and glycopeptide extraction, both at analytical and preparative scales, and available options for peptide detection with MS are discussed. Current algorithms for peptide sequence determination, and both analytical and computational techniques for quantification are compared. Techniques for statistical analysis, sequence mapping, enzyme prediction, and peptide function, and structure prediction are explored.

Dallas, D. C., et al. (2014). “Endogenous Human Milk Peptide Release Is Greater after Preterm Birth than Term Birth..” J Nutr.
BACKGROUND: Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile. OBJECTIVE: This study examines differences in peptides present between milk from women delivering at term and women delivering prematurely. METHODS: Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d). RESULTS: Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides. Conclusion: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant’s immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127.

Dallas, D. C., et al. (2014). “Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides” Food Res Int 63(Pt B): 203-209.
Whey permeate is a co-product obtained when cheese whey is passed through an ultrafiltration membrane to concentrate whey proteins. Whey proteins are retained by the membrane, whereas the low-molecular weight compounds such as lactose, salts, oligosaccharides and peptides pass through the membrane yielding whey permeate. Research shows that bovine milk from healthy cows contains hundreds of naturally occurring peptides – many of which are homologous with known antimicrobial and immunomodulatory peptides – and nearly 50 oligosaccharide compositions (not including structural isomers). As these endogenous peptides and oligosaccharides have low-molecular weight and whey permeate is currently an under-utilized product stream of the dairy industry, we hypothesized that whey permeate may serve as an inexpensive source of naturally occurring functional peptides and oligosaccharides. Laboratory fractionation of endogenous peptides and oligosaccharides from bovine colostrum sweet whey was expanded to pilot-scale. The membrane fractionation methodology used was similar to the methods commonly used industrially to produce whey protein concentrate and whey permeate. Pilot-scale fractionation was compared to laboratory-scale fractionation with regard to the identified peptides and oligosaccharide compositions. Results were interpreted on the basis of whether industrial whey permeate could eventually serve as a source of functional peptides and oligosaccharides. The majority (96%) of peptide sequences and the majority (96%) of oligosaccharide compositions found in the laboratory-scale process were mirrored in the pilot-scale process. Moreover, the pilot-scale process recovered an additional 33 peptides and 1 oligosaccharide not identified from the laboratory-scale extraction. Both laboratory- and pilot-scale processes yielded peptides deriving primarily from the protein beta-casein. The similarity of the laboratory-and pilot-scale’s resulting peptide and oligosaccharide profiles demonstrates that whey permeate can serve as an industrial-scale source of bovine milk peptides and oligosaccharides.

De Leoz, M. L., et al. (2014). “Human Milk Glycomics and Gut Microbial Genomics in Infant Feces Show a Correlation between Human Milk Oligosaccharides and Gut Microbiota- A Proof-of-Concept Study” J Proteome Res.
Human milk oligosaccharides (HMOs) play a key role in shaping and maintaining a healthy infant gut microbiota. This article demonstrates the potential of combining recent advances in glycomics and genomics to correlate abundances of fecal microbes and fecal HMOs. Serial fecal specimens from two healthy breast-fed infants were analyzed by bacterial DNA sequencing to characterize the microbiota and by mass spectrometry to determine abundances of specific HMOs that passed through the intestinal tract without being consumed by the luminal bacteria. In both infants, the fecal bacterial population shifted from non-HMO-consuming microbes to HMO-consuming bacteria during the first few weeks of life. An initial rise in fecal HMOs corresponded with bacterial populations composed primarily of non-HMO-consuming Enterobacteriaceae and Staphylococcaeae. This was followed by decreases in fecal HMOs as the proportion of HMO-consuming Bacteroidaceae and Bifidobacteriaceae increased. Analysis of HMO structures with isomer differentiation revealed that HMO consumption is highly structure-specific, with unique isomers being consumed and others passing through the gut unaltered. These results represent a proof-of-concept and are consistent with the highly selective, prebiotic effect of HMOs in shaping the gut microbiota in the first weeks of life. The analysis of selective fecal bacterial substrates as a measure of alterations in the gut microbiota may be a potential marker of dysbiosis.

Guerrero, A., et al. (2014). “Mechanistic peptidomics- factors that dictate specificity in the formation of endogenous peptides in human milk” Mol Cell Proteomics 13(12): 3343-3351.
An extensive mass spectrometry analysis of the human milk peptidome has revealed almost 700 endogenous peptides from 30 different proteins. Two in-house computational tools were created and used to visualize and interpret the data through both alignment of the peptide quasi-molecular ion intensities and estimation of the differential enzyme participation. These results reveal that the endogenous proteolytic activity in the mammary gland is remarkably specific and well conserved. Certain proteins-not necessarily the most abundant ones-are digested by the proteases present in milk, yielding endogenous peptides from selected regions. Our results strongly suggest that factors such as the presence of specific proteases, the position and concentration of cleavage sites, and, more important, the intrinsic disorder of segments of the protein drive this proteolytic specificity in the mammary gland. As a consequence of this selective hydrolysis, proteins that typically need to be cleaved at specific positions in order to exert their activity are properly digested, and bioactive peptides encoded in certain protein sequences are released. Proteins that must remain intact in order to maintain their activity in the mammary gland or in the neonatal gastrointestinal tract are unaffected by the hydrolytic environment present in milk. These results provide insight into the intrinsic structural mechanisms that facilitate the selectivity of the endogenous milk protease activity and might be useful to those studying the peptidomes of other biofluids.

Guerrero, A., et al. (2014). “Top-Down Analysis of Highly Post-Translationally Modified Peptides by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry” J Am Soc Mass Spectrom.
Bovine kappa-caseinoglycomacropeptide (GMP) is a highly modified peptide from kappa-casein produced during the cheese making process. The chemical nature of GMP makes analysis by traditional proteomic approaches difficult, as the peptide bears a strong net negative charge and a variety of post-translational modifications. In this work, we describe the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) for the top-down analysis of GMP. The method allows the simultaneous detection of different GMP forms that result from the combination of amino acid genetic variations and post-translational modifications, specifically phosphorylation and O-glycosylation. The different GMP forms were identified by high resolution mass spectrometry in both negative and positive mode and confirmation was achieved by tandem MS. The results showed the predominance of two genetic variants of GMP that occur as either mono- or bi-phosphorylated species. Additionally, these four forms can be modified with up to two O-glycans generally sialylated. The results demonstrate the presence of glycosylated, bi-phosphorylated forms of GMP never described before.

Holton, T. A., et al. (2014). “Following the digestion of milk proteins from mother to baby” J Proteome Res 13(12): 5777-5783.
Little is known about the digestive process in infants. In particular, the chronological activity of enzymes across the course of digestion in the infant remains largely unknown. To create a temporal picture of how milk proteins are digested, enzyme activity was compared between intact human milk samples from three mothers and the gastric samples from each of their 4-12 day postpartum infants, 2 h after breast milk ingestion. The activities of 7 distinct enzymes are predicted in the infant stomach based on their observed cleavage pattern in peptidomics data. We found that the same patterns of cleavage were evident in both intact human milk and gastric milk samples, demonstrating that the enzyme activities that begin in milk persist in the infant stomach. However, the extent of enzyme activity is found to vary greatly between the intact milk and gastric samples. Overall, we observe that milk-specific proteins are cleaved at higher levels in the stomach compared to human milk. Notably, the enzymes we predict here only explain 78% of the cleavages uniquely observed in the gastric samples, highlighting that further investigation of the specific enzyme activities associated with digestion in infants is warranted.

Maverakis, E., et al. (2015). “Glycans in the immune system and The Altered Glycan Theory of Autoimmunity- A critical review.” J Autoimmun.
Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode “self” identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses.

Mehra, R., et al. (2014). “Novel high-molecular weight fucosylated milk oligosaccharides identified in dairy streams.” PLoS One 9(5): e96040.
Oligosaccharides are the third largest component in human milk. This abundance is remarkable because oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant milk formula, the technologies to reproduce the variety and complexity of human milk oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable oligosaccharides. Recent research has demonstrated that bovine milk and whey permeate also contain oligosaccharides. Thus, a thorough characterization of oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine milk oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called “mother liquor”, and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant oligosaccharides present in human milk. Notably 6 oligosaccharides contained fucose, a sugar monomer that is highly abundant in human milk, but is rarely observed in bovine milk. This work shows that dairy streams represent a potential source of complex milk oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human milk.

Totten, S. M., et al. (2014). “Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies” Anal Bioanal Chem 406(30): 7925-7935.
Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Underwood, M. A., et al. (2015). “Bifidobacterium longum subspecies infantis: champion colonizer of the infant gut.” Pediatr Res 77(1-2): 229-235.
Oligosaccharides are abundant in human milk. Production of these highly diverse structures requires significant energy expenditure by the mother and yet these human milk oligosaccharides offer no direct nutritive value to her infant. A primary function of human milk oligosaccharides is to shape the infant’s intestinal microbiota with life-long consequences. Bifidobacterium longum subspecies infantis (B. infantis) is unique among gut bacteria in its prodigious capacity to digest and consume any human milk oligosaccharide structure, the result of a large repertoire of bacterial genes encoding an array of glycosidases and oligosaccharide transporters not found in other bacterial species. In vitro, B. infantis grows better than other bacterial strains in the presence of human milk oligosaccharides, displays anti-inflammatory activity in premature intestinal cells, and decreases intestinal permeability. In premature infants, B. infantis given in combination with human milk increases B. infantis and decreases Enterobacteriaceae in the feces. Probiotics containing B. infantis decrease the risk of necrotizing enterocolitis in premature infants. Colonization with B. infantis is also associated with increased vaccine responses. Probiotic organisms have historically been selected based on ease of production and stability. The advantages of B. infantis, selected through coevolution with human milk glycans, present an opportunity for focused manipulation of the infant intestinal microbiota.

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