O-linked glycosylations in human milk casein and major whey proteins during lactation

Martin Nørmark Thesbjerg*, Katrine Overgaard Poulsen, Julie Astono, Nina Aagaard Poulsen, Lotte Bach Larsen, Søren Drud-Heydary Nielsen, Allan Stensballe, Ulrik Kræmer Sundekilde

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearch


As glycosylations are difficult to analyze, their roles and effects are poorly understood. Glycosylations in human milk (HM) differ across lactation. Glycosylations can be involved in antimicrobial activities and may serve as food for beneficial microorganisms. This study aimed to identify and analyze O-linked glycans in HM by high-throughput mass spectrometry. 184 longitudinal HM samples from 66 donors from day 3 and months 1, 2, and 3 postpartum were subjected to a post-translational modification specific enrichment-based strategy using TiO 2 and ZrO 2 beads for O-linked glycopeptide enrichment. β-CN was found to be a major O-linked glycoprotein, additionally, α S1-CN, κ-CN, lactotransferrin, and albumin also contained O-linked glycans. As glycosyltransferases and glycosidases are involved in assembling the glycans including O-linked glycosylations, these were further investigated. Some glycosyltransferases and glycosidases were found to be significantly decreasing through lactation, including two O-linked glycan initiator enzymes (GLNT1 and GLNT2). Despite their decrease, the overall level of O-linked glycans remained stable in HM over lactation. Three different motifs for O-linked glycosylation were enriched in HM proteins: Gly-Xxx-Xxx-Gly-Ser/Thr, Arg-Ser/Thr and LysSer/Thr. Further O-linked glycan motifs on β-CN were observed to differ between intact proteins and endogenous peptides in HM.

Original languageEnglish
Article number131613
JournalInternational Journal of Biological Macromolecules
IssuePart 2
Number of pages11
Publication statusPublished - Apr 2024


  • Casein
  • Human milk
  • O-linked glycosylation
  • Whey protein
  • β-CN
  • Glycopeptides/metabolism
  • Humans
  • Whey Proteins/chemistry
  • Glycosylation
  • Polysaccharides/chemistry
  • Milk, Human/chemistry
  • Lactation/metabolism
  • Female
  • Protein Processing, Post-Translational
  • Caseins/metabolism


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