Analysis of Milk Oligosaccharides by Mass Spectrometry

  • Lauren D. Wu
  • L. Renee Ruhaak
  • Carlito B. LebrillaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1503)


Human milk oligosaccharides (HMOs) are a highly abundant constituent in human milk, and its protective and prebiotic properties have attracted considerable attention. HMOs have been shown to directly and indirectly benefit the overall health of the infant due to a number of functions including serving as a beneficial food for gut bacteria, block to pathogens, and aiding in brain development. Researchers are currently exploring whether these structures may act as possible disease and nutrition biomarkers. Because of this, rapid-throughput methods are desired to investigate biological activity in large patient sets. We have optimized a rapid-throughput protocol to analyze human milk oligosaccharides using micro-volumes of human breast milk for nutritional biomarkers. This method may additionally be applied to other biological fluid substrates such as plasma, urine, and feces. The protocol involves lipid separation via centrifugation, protein precipitation using ethanol, alditol reduction with sodium borohydride, and a final solid-phase extraction purification step using graphitized carbon cartridges. Samples are analyzed using HPLC-Chip/TOF-MS and data filtered on Agilent MassHunter using an in-house library. Individual structural identification is matched against a previously developed HMO library using accurate mass and retention time. Using this method will allow in-depth characterization and profiling of HMOs in large patient sets, and will ease the process of discovering significant nutritional biomarkers in human milk.

Key words

Oligosaccharide Human milk Mass spectrometry Glycan Carbohydrate Free oligosaccharide HPLC TOF 


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Lauren D. Wu
    • 1
  • L. Renee Ruhaak
    • 2
    • 3
  • Carlito B. Lebrilla
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of CaliforniaDavisUSA
  2. 2.Department of Translational Molecular PathologyUT MD Anderson Cancer CenterHoustonUSA
  3. 3.Department of ChemistryUC DavisDavisUSA

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