Lactation and neonatal nutrition: defining and refining the critical questions.

This paper resulted from a conference entitled "Lactation and Milk: Defining and refining the critical questions" held at the University of Colorado School of Medicine from January 18-20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research.

Endo-β-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins.

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source.

Multiple precursor ion scanning of gangliosides and sulfatides with a reversed-phase microfluidic chip and quadrupole time-of-flight mass spectrometry.

Precise profiling of polar lipids including gangliosides and sulfatides is a necessary step in understanding the diverse physiological role of these lipids. We have established an efficient method for the profiling of polar lipids using reversed-phase nano high-performance liquid chromatography microfluidic chip quadrupole time-of-flight mass spectrometry (nano-HPLC-chip Q-TOF/MS). A microfluidic chip design provides improved chromatographic performance, efficient separation, and stable nanospray while the advanced high-resolution mass spectrometer allowed for the identification of complex isobaric polar lipids such as NeuAc- and NeuGc-containing gangliosides.

Natural variability in bovine milk oligosaccharides from Danish Jersey and Holstein-Friesian breeds.

Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification.

Advances in analysis of human milk oligosaccharides.

Oligosaccharides in human milk strongly influence the composition of the gut microflora of neonates. Because it is now clear that the microflora play important roles in the development of the infant immune system, human milk oligosaccharides (HMO) are studied frequently. Milk samples contain complex mixtures of HMO, usually comprising several isomeric structures that can be either linear or branched.

Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry.

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip.

Identification and characterization of complex bioactive oligosaccharides in white and red wine by a combination of mass spectrometry and gas chromatography.

Over forty-five complex free oligosaccharides (of which several are novel) have been isolated and chemically characterized by gas chromatography and high resolution and high mass accuracy matrix-assisted laser desorption/ionization mass spectrometry (MALDI-FTICR MS) in red and white wines, Grignolino and Chardonnay, respectively. Oligosaccharides with a degree of polymerization between 3 and 14 were separated from simple monosaccharides and disaccharides by solid-phase extraction. The concentrations of free oligosaccharides were over 100 mg/L in both red and white wines.

Use of attenuated total reflectance Fourier transform infrared spectroscopy to study lactosylceramide and GD3 DMPC bilayers.

Attenuated total reflection Fourier transform infrared spectroscopy was used to monitor the formation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), DMPC: lactosylceramide, and DMPC: GD3 lipid bilayers onto a zinc selenide surface. Infrared absorption peak position, bandwidth, and intensity were all used to monitor the formation, acyl chain ordering, and chemical environment within each bilayer. The results from this study indicate that the addition of glycosphingolipids into a DMPC lipid bilayer introduces decreases in both, acyl chain ordering, and homogeneity within the bilayer.

Coupling flash liquid chromatography with mass spectrometry for enrichment and isolation of milk oligosaccharides for functional studies.

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories.

Annotation of a serum N-glycan library for rapid identification of structures.

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides.

Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation enhancing its role in pathogenic bacteria-host interactions.

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported.

Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells.

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures.

Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides.

Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect.

The influence of milk oligosaccharides on microbiota of infants: opportunities for formulas.

In addition to a nutritive role, human milk also guides the development of a protective intestinal microbiota in the infant. Human milk possesses an overabundance of complex oligosaccharides that are indigestible by the infant yet are consumed by microbial populations in the developing intestine. These oligosaccharides are believed to facilitate enrichment of a healthy infant gastrointestinal microbiota, often associated with bifidobacteria.

N-Glycan profiling of dried blood spots.

Serum N-glycan profiles for use as clinical biomarkers of disease(s) is of increasing scientific interest. Promising profiles have already been identified in several diseases, including cancer, Alzheimer's, and diabetes. Venipuncture is routinely performed to collect the blood necessary for this type of analysis, but blood from a fingerstick placed on filter paper (known as dried blood spots (DBS)) is more advantageous.

Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways.

Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs.

Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane.

Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers.

Glycosylation is highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Glycan compositional profiling of human serum with mass spectrometry has already identified potential biomarkers for several types of cancer and diseases; however, composition alone does not fully describe glycan stereo- and regioisomeric diversity. The vast structural heterogeneity of glycans presents a formidable analytical challenge.

Nano-LC-MS/MS of glycopeptides produced by nonspecific proteolysis enables rapid and extensive site-specific glycosylation determination.

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopeptides and the ability to determine glycan composition and site-specific glycosylation.

Structure elucidation of native N- and O-linked glycans by tandem mass spectrometry (tutorial).

Oligosaccharides play important roles in many biological processes. However, the structural elucidation of oligosaccharides remains a major challenge due to the complexities of their structures. Mass spectrometry provides a powerful method for determining oligosaccharide composition.

Site-specific protein glycosylation analysis with glycan isomer differentiation.

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system.

Transcriptome profiling of bovine milk oligosaccharide metabolism genes using RNA-sequencing.

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing.

Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.

Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining nonspecific protease digestion, nanoflow liquid chromatography, and tandem mass spectrometry (MS/MS) aimed at comprehensive site-specific glycosylation analysis in protein mixtures.

N-linked glycan profiling of mature human milk by high-performance microfluidic chip liquid chromatography time-of-flight tandem mass spectrometry.

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS.

An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides.

Lactating mothers secrete milk sialyloligosaccharides (MSOs) that function as anti-adhesives once provided to the neonate. Particular infant-associated commensals, such as Bifidobacterium longum subsp. infantis, consume neutral milk oligosaccharides, although their ability to utilize acidic oligosaccharides has not been assessed.