scholarly journals Comparison of Different Labeling Techniques for the LC-MS Profiling of Human Milk Oligosaccharides

2021 ◽  
Vol 9 ◽  
Author(s):  
Yinzhi Lang ◽  
Yongzhen Zhang ◽  
Chen Wang ◽  
Limei Huang ◽  
Xiaoxiao Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Milk ◽  
Biological Activities ◽  
Complex Mixture ◽  
Molecular Ions ◽  
Quantitative Detection ◽  
Human Milk Oligosaccharides ◽  
Combination Strategy ◽  
Milk Oligosaccharides ◽  
Chromatography Mass Spectrometry

Human milk oligosaccharides (HMOs) exhibit various biological activities for infants, such as serving as prebiotics, blocking pathogens, and aiding in brain development. HMOs are a complex mixture of hetero-oligosaccharides that are generally highly branched, containing multiple structural isomers and no intrinsic chromophores, presenting a challenge to both their resolution and quantitative detection. While liquid chromatography-mass spectrometry (LC-MS) has become the primary strategy for analysis of various compounds, the very polar and chromophore-free properties of native glycans hinder their separation in LC and ionization in MS. Various labeling approaches have been developed to achieve separation of glycans with higher resolution and greater sensitivity of detection. Here, we compared five commonly used labeling techniques [by 2-aminobenzamide, 2-aminopyridine, 2-aminobenzoic acid (2-AA), 2,6-diaminopyridine, and 1-phenyl-3-methyl-5-pyrazolone] for analyzing HMOs specifically under hydrophilic-interaction chromatography-mass spectrometry (HILIC-MS) conditions. The 2-AA labeling showed the most consistent deprotonated molecular ions, the enhanced sensitivity with the least structural selectivity, and the sequencing-informative tandem MS fragmentation spectra for the widest range of HMOs; therefore, this labeling technique was selected for further optimization under the porous graphitized carbon chromatography-mass spectrometry (PGC-MS) conditions. The combination strategy of 2-AA labeling and PGC-MS techniques provided online decontamination (removal of excess 2-AA, salts, and lactose) and resolute detection of many HMOs, enabling us to characterize the profiles of complicated HMO mixtures comprehensively in a simple protocol.

scholarly journals Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

2016 ◽  
Vol 82 (12) ◽  
pp. 3622-3630 ◽  
Author(s):  
Sercan Karav ◽  
Annabelle Le Parc ◽  
Juliana Maria Leite Nobrega de Moura Bell ◽  
Steven A. Frese ◽  
Nina Kirmiz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Milk ◽  
Bifidobacterium Longum ◽  
Bovine Milk ◽  
Selective Growth ◽  
Whey Protein Concentrate ◽  
Human Milk Oligosaccharides ◽  
Milk Oligosaccharides ◽  
Content Type ◽  
Growth Substrates

ABSTRACTMilk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intactN-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 releasedN-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-releasedN-glycans supported the rapid growth ofBifidobacterium longumsubsp.infantis(B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth ofBifidobacterium animalissubsp.lactis(B. lactis), a species which does not. Conversely,B. infantisATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed thatB. infantisconsumed 73% of neutral and 92% of sialylatedN-glycans, whileB. lactisdegraded only 11% of neutral and virtually no (<1%) sialylatedN-glycans. These results provide mechanistic support thatN-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, releasedN-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety ofN-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides.IMPORTANCEIt has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugatedN-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth ofB. infantis, which is a key infant gut microbe. The selectivity of consumption of individualN-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip–quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal thatB. infantiscan consume the range of glycan structures released from whey protein concentrate.

scholarly journals Human Milk Oligosaccharides Influence Neonatal Mucosal and Systemic Immunity

2016 ◽  
Vol 69 (Suppl. 2) ◽  
pp. 41-51 ◽  
Author(s):  
Sharon M. Donovan ◽  
Sarah S. Comstock
Keyword(s):  
Human Milk ◽  
Infant Formula ◽  
Bovine Milk ◽  
Complex Mixture ◽  
Structural Diversity ◽  
Host Cells ◽  
Human Milk Oligosaccharides ◽  
Milk Oligosaccharides ◽  
Immune Development ◽  
Breastfed Infants

The immune system of the infant is functionally immature and naïve. Human milk contains bioactive proteins, lipids, and carbohydrates that protect the newborn and stimulate innate and adaptive immune development. This review will focus on the role human milk oligosaccharides (HMO) play in neonatal gastrointestinal and systemic immune development and function. For the past decade, intense research has been directed at defining the complexity of oligosaccharides in the milk of many species and is beginning to delineate their diverse functions. These studies have shown that human milk contains a higher concentration as well as a greater structural diversity and degree of fucosylation than the milk oligosaccharides in other species, particularly bovine milk from which many infant formulae are produced. The commercial availability of large quantities of certain HMO has furthered our understanding of the functions of specific HMO, which include protecting the infant from pathogenic infections, facilitating the establishment of the gut microbiota, promoting intestinal development, and stimulating immune maturation. Many of these actions are exerted through carbohydrate-carbohydrate interactions with pathogens or host cells. Two HMOs, 2′-fucosyllactose (2′FL) and lacto-N-neotetraose (LNnT), have recently been added to infant formula. Although this is a first step in narrowing the compositional gap between human milk and infant formula, it is unclear whether 1 or 2 HMO will recapitulate the complexity of actions exerted by the complex mixture of HMO ingested by breastfed infants. Thus, as more HMO become commercially available, either isolated from bovine milk or chemically or microbially synthesized, it is anticipated that more oligosaccharides will be added to infant formula either alone or in combination with other prebiotics.

Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry

2007 ◽  
Vol 361 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Milady R. Ninonuevo ◽  
Robert E. Ward ◽  
Riccardo G. LoCascio ◽  
J. Bruce German ◽  
Samara L. Freeman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Milk ◽  
Human Milk Oligosaccharides ◽  
Milk Oligosaccharides ◽  
Bacterial Fermentation

scholarly journals Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3482 ◽  
Author(s):  
Fischöder ◽  
Cajic ◽  
Grote ◽  
Heinzler ◽  
Reichl ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Human Milk ◽  
Absolute Quantification ◽  
Human Milk Oligosaccharides ◽  
Milk Oligosaccharides ◽  
Enzymatic Production ◽  
Flight Mass Spectrometry ◽  
Enzymatic Cascades ◽  
Biological Mixtures

Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple "routine" method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [13C6]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[15N]GlcNAc and its application for the enzymatic synthesis of [13C6/15N]lacto-N-neo-tetraose for the first time. An exemplary application was selected—analysis of tetraose in complex biological mixtures—to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.

scholarly journals Cryogenic IR spectroscopy combined with ion mobility spectrometry for the analysis of human milk oligosaccharides

The Analyst ◽  
2018 ◽  
Vol 143 (8) ◽  
pp. 1846-1852 ◽  
Author(s):  
Neelam Khanal ◽  
Chiara Masellis ◽  
Michael Z. Kamrath ◽  
David E. Clemmer ◽  
Thomas R. Rizzo
Keyword(s):  
Mass Spectrometry ◽  
Infrared Spectroscopy ◽  
Ir Spectroscopy ◽  
Human Milk ◽  
Ion Mobility Spectrometry ◽  
Ion Mobility ◽  
Human Milk Oligosaccharides ◽  
Milk Oligosaccharides

Cryogenic, messenger-tagging, infrared spectroscopy is combined with ion mobility spectrometry and mass spectrometry to identify isomeric human milk oligosaccharides (HMOs) ranging from trisaccharides to hexasaccharides.

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