Comparison of the human and bovine milk N-Glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry

Charles C. Nwosu, Danielle L. Aldredge, Hyeyoung Lee, Larry A. Lerno, Angela M. Zivkovic, J. Bruce German, Carlito B Lebrilla

Research output: Contribution to journalArticle

96 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2912-2924
Number of pages13
JournalJournal of Proteome Research
Volume11
Issue number5
DOIs
StatePublished - May 4 2012

Fingerprint

Microfluidics
Liquid chromatography
Human Milk
Tandem Mass Spectrometry
Liquid Chromatography
Mass spectrometry
Polysaccharides
Milk
Mannose
Milk Proteins
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Glycosylation
Oligosaccharides
Proteins
Centrifugation
Mass Spectrometry
Chemical analysis
Ethanol
Carbon
Isomers

Keywords

  • human and bovine milk
  • N-glycans
  • nano-LC?Q-TOF MS
  • NeuGc
  • PGC Chip
  • PNGase F
  • tandem MS

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Nwosu, C. C., Aldredge, D. L., Lee, H., Lerno, L. A., Zivkovic, A. M., German, J. B., & Lebrilla, C. B. (2012). Comparison of the human and bovine milk N-Glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry. Journal of Proteome Research, 11(5), 2912-2924. https://doi.org/10.1021/pr300008u

Comparison of the human and bovine milk N-Glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry. / Nwosu, Charles C.; Aldredge, Danielle L.; Lee, Hyeyoung; Lerno, Larry A.; Zivkovic, Angela M.; German, J. Bruce; Lebrilla, Carlito B.

In: Journal of Proteome Research, Vol. 11, No. 5, 04.05.2012, p. 2912-2924.

Research output: Contribution to journalArticle

Nwosu, Charles C. ; Aldredge, Danielle L. ; Lee, Hyeyoung ; Lerno, Larry A. ; Zivkovic, Angela M. ; German, J. Bruce ; Lebrilla, Carlito B. / Comparison of the human and bovine milk N-Glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry. In: Journal of Proteome Research. 2012 ; Vol. 11, No. 5. pp. 2912-2924.
@article{538bc817bfcb4c8f9ac6c632684b570c,
title = "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.",
keywords = "human and bovine milk, N-glycans, nano-LC?Q-TOF MS, NeuGc, PGC Chip, PNGase F, tandem MS",
author = "Nwosu, {Charles C.} and Aldredge, {Danielle L.} and Hyeyoung Lee and Lerno, {Larry A.} and Zivkovic, {Angela M.} and German, {J. Bruce} and Lebrilla, {Carlito B}",
year = "2012",
month = "5",
day = "4",
doi = "10.1021/pr300008u",
language = "English (US)",
volume = "11",
pages = "2912--2924",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "5",

}

TY - JOUR

T1 - Comparison of the human and bovine milk N-Glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry

AU - Nwosu, Charles C.

AU - Aldredge, Danielle L.

AU - Lee, Hyeyoung

AU - Lerno, Larry A.

AU - Zivkovic, Angela M.

AU - German, J. Bruce

AU - Lebrilla, Carlito B

PY - 2012/5/4

Y1 - 2012/5/4

N2 - 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.

AB - 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.

KW - human and bovine milk

KW - N-glycans

KW - nano-LC?Q-TOF MS

KW - NeuGc

KW - PGC Chip

KW - PNGase F

KW - tandem MS

UR - http://www.scopus.com/inward/record.url?scp=84860626670&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84860626670&partnerID=8YFLogxK

U2 - 10.1021/pr300008u

DO - 10.1021/pr300008u

M3 - Article

VL - 11

SP - 2912

EP - 2924

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 5

ER -