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Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes.

Authors
  • Zhu, Jing1, 2, 3
  • Lin, Yu-Hsien1, 2
  • Dingess, Kelly A1, 2
  • Mank, Marko4
  • Stahl, Bernd4, 5
  • Heck, Albert J R1, 2
  • 1 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. , (Netherlands)
  • 2 Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands. , (Netherlands)
  • 3 Beijing Institute of Nutritional Resources, 100069 Beijing, China. , (China)
  • 4 Danone Nutricia Research, Uppsalalaan 12, 3584 CT Utrecht, The Netherlands. , (Netherlands)
  • 5 Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CG Utrecht, The Netherlands. , (Netherlands)
Type
Published Article
Journal
Journal of Proteome Research
Publisher
American Chemical Society
Publication Date
Mar 27, 2020
Identifiers
DOI: 10.1021/acs.jproteome.9b00753
PMID: 32125861
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Protein N-glycosylation on human milk proteins assists in protecting an infant's health and functions among others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother's milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein- and N-site-specific quantitative information. Here, we describe an automated platform using hydrophilic-interaction chromatography (HILIC)-based cartridges enabling the proteome-wide monitoring of intact N-glycopeptides using just a digest of 150 μg of breast milk protein. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. Although each glycoprotein, N-glycosylation site, and attached glycan revealed distinct dynamic changes, we did observe a few general trends. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Building on the improved glycoproteomics approach outlined above, future studies are warranted to reveal the potential impact of the observed glycosylation microheterogeneity on the healthy development of infants.

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