Annotation and structural elucidation of bovine milk oligosaccharides and determination of novel fucosylated structures

Abstract

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structure-related specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomer-specific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.

Keywords: bovine colostrum; high-performance liquid chromatography; oligosaccharides; tandem mass spectrometry.

Figures

Fig. 1.

(A) The MALDI FT-ICR MS profile of a reduced BMO pool in the positive-ion mode. The numbers shown above the peaks represent, from bottom to top, the number of Hex, HexNAc, Fuc and NeuAc residues. Most ions are [M+Na]+ and the [M-H+2Na]+ ions are denoted with a star. (B) The -HPLC-Chip/TOF MS profile of the reduced BMO pool. Major peaks are labeled with putative structures, where blue circles denote Glc, blue squares denote GlcNAc, yellow circles denote Gal, purple diamonds denote NeuAc and red triangles denote Fuc. (These symbols are used throughout all figures.)

Fig. 2.

(A) MALDI FT-ICR MS profile of fraction 28 (from the off-line BMO pool separation) showing the isolation of m/z 1097.4 with EIC inset. (B) MS profile of fraction 28 after digestion with β1-4 galactosidase showing loss of two hexose mass shift with the EIC inset.

Fig. 3.

(A) EIC of m/z 507.2 from the BMO pool showing four isomers with their respective structures labeled. (B) EIC of m/z 507.2 at the half-way point of digestion with β1-3,6 galactosidase. (C) EIC of m/z 507.2 after full-time digestion with β1-3,6 galactosidase. (D) EIC of m/z 507.2 after digestion with β1-4 galactosidase. (E) EIC of m/z 710.2 from the BMO pool showing seven isomers. (F) EIC of m/z 710.2 after digestion with β1-4 galactosidase.

Fig. 4.

(A) EIC of m/z 548.2 from the BMO pool showing two main isomers. (B) EIC of m/z 548.2 after digestion with α N-acetylgalactosaminidase. (C) EIC of m/z 548.2 after digestion with β N-acetylhexosaminidase. (D) Magnification on base line of m/z 548.2 from the BMO pool showing additional isomers. (E) Isomer-specific fragmentation of EIC m/z 548.2 at 12.5 min, confirming reducing end HexNAc.

Fig. 5.

(A) Isomer-specific fragmentation profile of EIC m/z 751.3 from the BMO pool at 14.8 min, with the putative structure inset. (B) Isomer-specific fragmentation profile of EIC m/z 751.3 from the BMO pool at 14.1 min, with the putative structure inset. (C) Isomer-specific fragmentation profile of EIC m/z 491.19 from the BMO pool at 12.6 min, with the structure and EIC inset.

Fig. 6.

(A) Isomer-specific fragmentation of EIC m/z 694.3 at 10.4 min, with the putative structure inset. (B) Isomer-specific fragmentation of EIC m/z 1018.4 at 14.3 min, with the putative structure inset.

Fig. 7.

(A) Isomer-specific fragmentation of EIC m/z 1059.4 at 13.8 min, with the putative structure inset. (B) BPC of the BMO pool. (C) BPC of the HMO pool. (D) EIC of m/z 507.2 from the BMO pool showing four isomers. (E) EIC of m/z 507.2 from the HMO pool showing two isomers.