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

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.

Challenges in Separations of Proteins and Small Biomolecules and the Role of Modern Mass Spectroscopy Tools for Solving Them, as Well as Bypassing Them, in Structural Analytical Studies of Complex Biomolecular Mixtures

“…In Figure 16, extracted compound chromatograms show elution profiles of human (Figure 16a) and bovine (Figure 16b) milk N-glycans, with putative glycan structures assigned [79]. In this example 38 N-glycan compositions were assigned for the human milk N-glycan pool, while 51 Nglycan compositions were assigned for the bovine one [79]. Taking into account isomeric structures, over a hundred compounds were present in both mixtures [79].…”

“…In both cases, bioinformatics is crucial for data analysis, as will be discussed later. For LC-MS analyses, porous graphitized carbon (PGC) chromatography represents again the method of choice for separation of N-glycans [79].…”

“…In this example 38 N-glycan compositions were assigned for the human milk N-glycan pool, while 51 N-glycan compositions were assigned for the bovine one [79]. Taking into account isomeric structures, over a hundred compounds were present in both mixtures [79].…”

“…In both cases, bioinformatics is crucial for data analysis, as will be discussed later. For LC-MS analyses, porous graphitized carbon (PGC) chromatography represents again the method of choice for separation of N-glycans [79]. In Figure 16, extracted compound chromatograms show elution profiles of human (Figure 16a) and bovine (Figure 16b) milk N-glycans, with putative glycan structures assigned [79].…”

“…For LC-MS analyses, porous graphitized carbon (PGC) chromatography represents again the method of choice for separation of N-glycans [79]. In Figure 16, extracted compound chromatograms show elution profiles of human (Figure 16a) and bovine (Figure 16b) milk N-glycans, with putative glycan structures assigned [79]. In this example 38 N-glycan compositions were assigned for the human milk N-glycan pool, while 51 Nglycan compositions were assigned for the bovine one [79].…”