David C. Dallas

Consumption of human milk glycoconjugates by infant-associated bifidobacteria: mechanisms and implications

Human milk is a rich source of nutrients and energy, shaped by mammalian evolution to provide all the nutritive requirements of the newborn. In addition, several molecules in breast milk act as bioactive agents, playing an important role in infant protection and guiding a proper development. While major breast milk nutrients such as lactose, lipids and proteins are readily digested and consumed by the infant, other molecules, such as human milk oligosaccharides and glycosylated proteins and lipids, can escape intestinal digestion and transit through the gastrointestinal tract. In this environment, these molecules guide the composition of the developing infant intestinal microbiota by preventing the colonization of enteric pathogens and providing carbon and nitrogen sources for other colonic commensals. Only a few bacteria, in particular Bifidobacterium species, can gain access to the energetic content of milk as it is displayed in the colon, probably contributing to their predominance in the intestinal microbiota in the first year of life. Bifidobacteria deploy exquisite molecular mechanisms to utilize human milk oligosaccharides, and recent evidence indicates that their activities also target other human milk glycoconjugates. Here, we review advances in our understanding of how these microbes have been shaped by breast milk components and the strategies associated with their consumption of milk glycoconjugates.

Current peptidomics: applications, purification, identification, quantification, and functional analysis.

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.

Extensive in vivo Human Milk Peptidomics Reveals Specific Proteolysis Yielding Protective Antimicrobial Peptides

Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin, and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective-released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus . The predigestion of milk proteins and the consequent release of antibacterial peptides may provide a selective advantage through evolution by protecting both the mothers mammary gland and her nursing offspring from infection.

Automated glycopeptide analysis—review of current state and future directions

Glycosylation of proteins is involved in immune defense, cell-cell adhesion, cellular recognition and pathogen binding and is one of the most common and complex post-translational modifications. Science is still struggling to assign detailed mechanisms and functions to this form of conjugation. Even the structural analysis of glycoproteins-glycoproteomics-remains in its infancy due to the scarcity of high-throughput analytical platforms capable of determining glycopeptide composition and structure, especially platforms for complex biological mixtures. Glycopeptide composition and structure can be determined with high mass-accuracy mass spectrometry, particularly when combined with chromatographic separation, but the sheer volume of generated data necessitates computational software for interpretation. This review discusses the current state of glycopeptide assignment software-advances made to date and issues that remain to be addressed. The various software and algorithms developed so far provide important insights into glycoproteomics. However, there is currently no freely available software that can analyze spectral data in batch and unambiguously determine glycopeptide compositions for N- and O-linked glycopeptides from relevant biological sources such as human milk and serum. Few programs are capable of aiding in structural determination of the glycan component. To significantly advance the field of glycoproteomics, analytical software and algorithms are required that: (i) solve for both N- and O-linked glycopeptide compositions, structures and glycosites in biological mixtures; (ii) are high-throughput and process data in batches; (iii) can interpret mass spectral data from a variety of sources and (iv) are open source and freely available.

Digestion of Protein in Premature and Term Infants.

Premature birth rates and premature infant morbidity remain discouragingly high. Improving nourishment for these infants is the key for accelerating their development and decreasing disease risk. Dietary protein is essential for growth and development of infants. Studies on protein nourishment for premature infants have focused on protein requirements for catch-up growth, nitrogen balance, and digestive protease concentrations and activities. However, little is known about the processes and products of protein digestion in the premature infant. This review briefly summarizes the protein requirements of term and preterm infants, and the protein content of milk from women delivering preterm and at term. An in-depth review is presented of the current knowledge of term and preterm infant dietary protein digestion, including human milk protease and anti-protease concentrations; neonatal intestinal pH, and enzyme activities and concentrations; and protein fermentation by intestinal bacteria. The advantages and disadvantages of incomplete protein digestion as well as factors that increase resistance to proteolysis of particular proteins are discussed. In order to better understand protein digestion in preterm and term infants, future studies should examine protein and peptide fragment products of digestion in saliva, gastric, intestinal and fecal samples, as well as the effects of the gut micro biome on protein degradation. The confluence of new mass spectrometry technology and new bioinformatics programs will now allow thorough identification of the array of peptides produced in the infant as they are digested.

Nutrigenomics and Personalized Diets: What Will They Mean for Food?

The modern food system feeds six billion people with remarkable diversity, safety, and nutrition. Yet, the current rise in diet-related diseases is compromising health and devaluing many aspects of modern agriculture. Steps to increase the nutritional quality of individual foods will assist in personalizing health and in guiding individuals to achieve superior health. Nutrigenomics is the scientific field of the genetic basis for varying susceptibilities to disease and the diverse responses to foods. Although some of these genetic determinants will be simple and amenable to personal genotyping as the means to predict health, in practice most will not. As a result, genotyping will not be the secret to personalizing diet and health. Human assessment technologies from imaging to proteomics and metabolomics are providing tools to both understand and accurately assess the nutritional phenotype of individuals. The business models are also emerging to bring these assessment capabilities to industrial practice, in which consumers will know more about their personal health and seek personal solutions.

A Peptidomic Analysis of Human Milk Digestion in the Infant Stomach Reveals Protein-Specific Degradation Patterns

In vitro digestion of isolated milk proteins results in milk peptides with a variety of actions. However, it remains unclear to what degree protein degradation occurs in vivo in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray LC separation with time-of-flight mass spectrometry, comprehensive structural libraries, and informatics to analyze milk from 3 human mothers and the gastric aspirates from their 4- to 12-d-old postpartum infants. Milk from the mothers contained almost 200 distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. Most peptides in both the intact milk and gastric samples were derived from β-casein. The numbers of peptides from β-casein, lactoferrin, α-lactalbumin, lactadherin, κ-casein, serum albumin, bile salt-associated lipase, and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than in the milk samples (P < 0.05). A total of 603 peptides differed significantly in abundance between milk and gastric samples (P < 0.05). Most of the identified peptides have previously identified biologic activity. Gastric proteolysis occurs in the term infant in the first 2 wk of life, releasing biologically active milk peptides with immunomodulatory and antibacterial properties of clinical relevance to the proximal intestinal tract. Data are available via ProteomeXchange (identifier PXD000688).

Mechanistic Peptidomics: Factors That Dictate Specificity in the Formation of Endogenous Peptides in Human Milk

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.

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. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.

Characterization of goat milk lactoferrin N-glycans and comparison with the N-glycomes of human and bovine milk.

Numerous milk components, such as lactoferrin, are recognized as health‐promoting compounds. A growing body of evidence suggests that glycans could mediate lactoferrins bioactivity. Goat milk lactoferrin is a candidate for infant formula supplementation because of its high homology with its human counterpart. The aim of this study was to characterize the glycosylation pattern of goat milk lactoferrin. After the protein was isolated from milk by affinity chromatography, N‐glycans were enzymatically released and a complete characterization of glycan composition was carried out by advanced MS. The glycosylation of goat milk lactoferrin was compared with that of human and bovine milk glycoproteins. Nano‐LC‐Chip‐Q‐TOF MS data identified 65 structures, including high mannose, hybrid, and complex N‐glycans. Among the N‐glycan compositions, 37% were sialylated and 34% were fucosylated. The results demonstrated the existence of similar glycans in human and goat milk but also identified novel glycans in goat milk that were not present in human milk. These data suggest that goat milk could be a source of bioactive compounds, including lactoferrin that could be used as functional ingredients for food products beneficial to human nutrition.