Annotation and curation of the first goat milk oligosaccharides library: from analytical characterization to health benefits

Project:

Annotation and curation of the first goat milk oligosaccharides library: from analytical characterization to health benefits

Daniela Barile

Free oligosaccharides are biomolecules that are present in human milk
and provide benefits beyond essential nutrients. Numerous functional
studies on human milk oligosaccharides (HMO) have concluded that these
molecules perform important biological functions including the
establishment of intestinal microbiota and prevention of pathogen
binding to intestinal cells. Mammalian free milk oligosaccharides are
commonly divided in two main groups based on their composition: neutral
and acidic. Neutral oligosaccharides are composed of galactose (Gal),
Glucose (Glc) N-Acetyl-glucosamine (GlcNAc), fucose (Fuc) and a
lactose core; acidic oligosaccharides are characterized by these same
monomers but also one molecule of N-acetyl-neuraminic acid (NeuAc) and N-glycolyl
neuraminic (NeuGc) are found. At present, the only source of milk
oligosaccharides is human milk, which limits oligosaccharides research
and practical applications. Because all the attempts to synthesize these
molecules have failed to mimic the complexity and bioactivities of
HMOs, much simpler structures are being used to supplement dietary
products and infant formula. Recent studies have documented that these
complex oligosaccharides are present in other mammalian milks, such as
goat and bovine milk. This project constitutes the first comprehensive
effort to profile and standardize the chemical structures and
nomenclature of oligosaccharides present in goat milk (GMO).

Identification of free oligosaccharides by mass spectrometry requires
that they are separated from the other components present in the
mixture. For the detailed methods please refer to Barile et al.,
International Dairy Journal (2009) PMID: 20161544 [PubMed] (link).

This library was constructed using an Agilent 6500 Series
HPLC-Chip/Quadrupole TOF Mass Spectrometer system equipped with
microfluidic Chip, HPLC-Chip interface, microwell-plate autosampler,
capillary sample loading pump and nanopump and with a Matrix-Assisted
Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry (MALDI FT-ICR MS).

The library includes the theoretical masses as measured by MALDI FT-ICR
and by Nano LC chip Q ToF as well as the universal neutral masses
(reduced and unreduced). The use of different technology platforms and
methods that do not require specific derivatization, is intended to
facilitate the access and exchange of experimental research activities
to develop and improve the fields of milk bioactives by
multidisciplinary cooperation. The library also contains the
conventional names, chemical structure, the monomeric composition,
reproducible chromatographic details and related articles in pdf. Unique
tandem (MS/MS) mass spec fingerprint generated by Q ToF are also
available.

Because milk oligosaccharides show extreme diversity in structure and
may contain various functional groups, they differ in bioactivity. Some
oligosaccharides are particularly interesting regarding their specific
activity in promoting the human health. In particular, fucosylated and
sialylated oligosaccharides are of special interest because they provide
the greatest biological activities. The addition of the functional
residues sialic acid and fucose to the oligosaccharide backbone is
dependent on the activity of specific enzymes excreted by the mammary
gland. Several sialyltransferases may add NeuAc units, producing acidic
oligosaccharides. The fucosylation of oligosaccharides is dependent on
enzymes encoded by the same genes that encode Lewis blood group types.
Neutral fucosylated oligosaccharides have been shown to inhibit adhesion
of other pathogens (i.e. Campylobacter jejuni, diarrheagenic E. coli) that
cause gastrointestinal disorders. Both fucosylated and neutral
oligosaccharides thus have the potential to inhibit infection caused by
enterotoxigenic and uropathogenic E. coli. Isolated acidic oligosaccharides (containing sialic acid) have been reported to block adhesion of Helicobacter pylori,
which causes peptic ulcers and other gastric diseases. The monomeric
composition of the oligosaccharides annotated in the library revealed
that most of goat milk oligosaccharides have a lactose core and they are
further decorated by functional groups. Seven fucosylated
oligosaccharides, 11 sialylated oligosaccharides with N-Acetylneuraminic acid and 6 sialylated oligosaccharides with N-glycolylneuraminic acid, and 18 oligosaccharides also containing HexNAc are described in the library.

View a base peak chromatogram (BPC) profile and an annotated BPC of goat milk oligosaccharides

List of abbreviations:

  • TOF, time-of-flight

  • BPC, base peak chromatogram

  • m/z, mass-to-charge ratio

  • Hex, hexose (glucose or galactose)

  • HexNAc, N-acetylhexosamine (N-acetylglucosamine or N-acetylgalactosamine)

  • NeuAc, N-acetyl neuraminic acid or sialic acid

  • NeuGc, N-glycolylneuraminic acid

  • Fuc, Fucose

Notes

The oligosaccharides ID in the library are used to refer to specific oligosaccharides for their tandem MS/MS fingerprints and structures and were obtained using their sequential monomeric composition. For example, the oligosaccharide with ID 11100 is made of 1 hexose + 1 HexNAc + 1 fucose + 0 NeuAc + 0 NeuGC.

The literature references are complete articles in pdf format and are
alphabetically ordered and linked to the specific oligosaccharide in the
library.