N-Linked Glycan Profiling of Mature Human Milk by High-Performance Microfluidic Chip Liquid Chromatography Time-of-Flight Tandem Mass Spectrometry

Abstract

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, be at least two-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 comprised 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

“…Data analysis revealed that goat milk lactoferrin N ‐glycans contained NeuAc or NeuGc. Both NeuAc‐ and NeuGc‐containing N ‐glycans are present in bovine milk proteins, but NeuGc‐containing N ‐glycans are not present in human milk proteins . In some instances, accurate mass alone is not sufficient to positively identify N ‐glycan compositions.…”

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

“…1); and (c) the hybrid type, which consists of a mixture of these two types. The human milk Nglycome has been recently described in detail, and in general human milk N-glycans are largely fucosylated and present in larger concentrations compared to bovine milk (Dallas et al, 2011;Nwosu et al, 2012). In contrast, bovine N-glycans are also more sialylated and characterized by the presence of N-glycolylneuraminic acid (NeuGc) instead of N-acetylneuraminic acid (Nwosu et al, 2012).…”

Structural Glycomic Analyses at High Sensitivity: A Decade of Progress

“…This combination demonstrated that several glycans were differentially present in the different states of health. LC chips with graphite packings are also available and have been used to characterize human milk oligosaccharides (118-120), milk carbohydrates from different primates (121), and glycans present on serum glycoproteins (122). Nearly 200 structures were identified from their exact masses in human serum; these structures included isomeric analytes (123), which were then used to construct a library based on m/z values and retention times to quickly determine the structure of a given glycan.…”