Analysis of Milk Oligosaccharides by Mass Spectrometry

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.

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Variations in bovine milk oligosaccharides during early and middle lactation stages analyzed by high-performance liquid chromatography-chip/mass spectrometry

Journal of Dairy Science ◽

10.3168/jds.2008-1642 ◽

2009 ◽

Vol 92 (7) ◽

pp. 2991-3001 ◽

Cited By ~ 110

Author(s):

N. Tao ◽

E.J. DePeters ◽

J.B. German ◽

R. Grimm ◽

C.B. Lebrilla

Keyword(s):

Mass Spectrometry ◽

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

High Performance ◽

Bovine Milk ◽

Milk Oligosaccharides

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

Frontiers in Chemistry ◽

10.3389/fchem.2021.691299 ◽

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.

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