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 and be at least 2-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 composed of N-glycans found in all three milk samples.

Figures

Figure 1

N-glycan identification strategy. The protein sample is divided into two equal parts and to one is added PNGase F, while the other is left unaltered as a control. The remaining experimental steps are run in parallel on both samples. N-glycans are compounds found at levels greater than or equal to two times higher in the PNGase F-incubated samples compared to the control samples.

Figure 2

Extracted Compound Chromatogram (ECC) for top N-glycans in the milk of donor A.

Figure 3

A: ECC's for 5 Hex 4 HexNAc and 1 dHex (894.33 m/z) in the N-glycan extracts of donor milk A. B-D: Fragmentation spectra for three isomers at 14.706 min (B), 15.163 min (C) and 16.059 min (D).

Figure 4. Total number of N-glycans in three different individual mothers' milks