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Comparative Study
. 2012 May 4;11(5):2912-24.
doi: 10.1021/pr300008u. Epub 2012 Apr 6.

Comparison of the Human and Bovine Milk N-glycome via High-Performance Microfluidic Chip Liquid Chromatography and Tandem Mass Spectrometry

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Free PMC article
Comparative Study

Comparison of the Human and Bovine Milk N-glycome via High-Performance Microfluidic Chip Liquid Chromatography and Tandem Mass Spectrometry

Charles C Nwosu et al. J Proteome Res. .
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Abstract

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.

Figures

Figure 1
Figure 1
(A) Extracted compound chromatogram (ECC) showing the elution profile of human milk N-glycans via nano-LC/MS. (B) Extracted compound chromatogram (ECC) showing the elution profile of bovine milk N-glycans via nano-LC/MS. Green circles ( formula image), yellow circles ( formula image), blue squares ( formula image), red triangles ( formula image), purple diamonds ( formula image) and grey diamonds ( formula image) represent mannose, galactose, GlcNAc, fucose, NeuAc and NeuGc residues, respectively.
Figure 2
Figure 2
Deconvoluted CID data for (A) a high mannose N-glycan with m/z 1235.45 corresponding in mass to [GlcNAc2+Man5+H]+1. (B) a high mannose N-glycan with m/z 942.33 corresponding in mass to [GlcNAc2+Man9+2H]+2. Both glycans represented in Figures 2A and 2B were observed in human and bovine milk.
Figure 3
Figure 3
Deconvoluted CID data for (A) a neutral bi-antennary complex N-glycan with m/z 821.31 corresponding in mass to [GlcNAc4+Hex5+2H]+2. (B) a neutral bi-antennary complex N-glycan with m/z 894.34 corresponding in mass to [GlcNAc4+Hex5+Fuc1+2H]+2. (C) a neutral bi-antennary complex N-glycan with m/z 862.34 corresponding in mass to [HexNAc6+Man3+2H]+2. Both glycans represented in Figures 3A and 3B were observed in human and bovine milk while the glycan represented in Figure 3C was unique to only the bovine milk source.
Figure 4
Figure 4
Deconvoluted CID data for (A) a NeuAc-sialylated N-glycan with m/z 966.86 corresponding in mass to [GlcNAc4+Hex5+NeuAc1+2H]+2. (B) a NeuGc-sialylated N-glycan with m/z 974.85 corresponding in mass to [GlcNAc4+Hex5+NeuGc1+2H]+2. (C) a bi-sialylated N-glycan with m/z 1120.40 corresponding in mass to [GlcNAc4+Hex5+NeuAc1+NeuGc1+2H]+2. The numerous unassigned peaks in Figure 4C are probably due to isobaric interferences in isolating the precursor ion. The glycan represented in Figure 4A was observed in both the human and bovine milk sources while the glycans represented in Figures 4B and 4C were unique to only the bovine milk source.
Figure 5
Figure 5
(A) Extracted ion chromatogram (EIC) showing two isomers for the N-glycan with m/z 813.31 corresponding in mass to [GlcNAc4+Hex4+Fuc1+2H]+2. (B) Deconvoluted CID data for the isomer eluting at 19.35 min. (C) Deconvoluted CID data for the isomer eluting at 20.00 min. Both glycans represented in Figures 5B and 5C were observed in human and bovine milk. The red bold arrows correspond to diagnostic ions that signify core and antenna fucosylation.
Figure 6
Figure 6
Pie charts showing the relative abundances of (A) all the N-glycan types in human milk. (B) fucosylated N-glycans in human milk. (C) all the N-glycan types in bovine milk. (D) fucosylated N-glycans in bovine milk.

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